Hepatocyte growth factor (HGF) binding proteins

ABSTRACT

The present invention provides a family of binding proteins that bind and neutralize the activity of hepatocyte growth factor (HGF), in particular human HGF. The binding proteins can be used as diagnostic and/or therapeutic agents. With regard to their therapeutic activity, the binding proteins can be used to treat certain HGF responsive disorders, for example, certain HGF responsive tumors.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/051,474, filed Mar. 18, 2011, now U.S. Pat. No. 8,268,315, which is adivisional of U.S. patent application Ser. No. 12/632,758, filed Dec. 7,2009, now U.S. Pat. No. 7,935,502, which is a divisional of U.S. patentapplication Ser. No. 11/757,094, filed Jun. 1, 2007, now U.S. Pat. No.7,659,378, which claims the benefit of and priority to U.S. ProvisionalApplication Nos. 60/810,714, filed Jun. 2, 2006, and 60/860,461, filedNov. 21, 2006, the entire disclosures of which are incorporated byreference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 28, 2013, isnamed AVO-001ADV3_SL.txt and is 293,557 bytes in size.

FIELD OF THE INVENTION

The field of the invention is molecular biology, immunology andoncology. More particularly, the field is antibody-based bindingproteins that bind human hepatocyte growth factor (HGF).

BACKGROUND

Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF), is amulti-functional heterodimeric protein produced predominantly bymesenchymal cells, and is an effector of cells expressing the Mettyrosine kinase receptor (Bottaro et al. (1991) SCIENCE 251: 802-804,Rubin et al. (1993) BIOCHIM. BIOPHYS. ACTA 1155: 357-371). The human Metreceptor is also known as “c-Met.” Mature HGF contains two polypeptidechains, the α-chain and the β-chain. Published studies suggest it is theα-chain that contains HGF's c-Met receptor binding domain.

When it binds to its cognate receptor, HGF mediates a number of cellularactivities. The HGF-Met signaling pathway plays a role in liverregeneration, wound healing, neural regeneration, angiogenesis andmalignancies. See, e.g., Cao et al. (2001) PROC. NATL. ACAD. SCI. USA98: 7443-7448, Burgess et al. (2006) CANCER RES. 66: 1721-1729, and U.S.Pat. Nos. 5,997,868 and 5,707,624. Investigators have been developing anumber of HGF modulators, including antibodies, to treat variousdisorders that involve HGF activity, for example, certain HGF responsivecancers. See, e.g., International Application Publication No. WO2005/017107.

The basic structure common to all antibodies is shown schematically inFIG. 1. Antibodies are multimeric proteins that contain four polypeptidechains. Two of the polypeptide chains are called heavy or H chains andtwo of the polypeptide chains are called light or L chains. Theimmunoglobulin heavy and light chains are connected by an interchaindisulfide bond. The immunoglobulin heavy chains are connected by anumber of interchain disulfide bonds. A light chain is composed of onevariable region (V_(L) in FIG. 1) and one constant region (C_(L) in FIG.1), while the heavy chain is composed of one variable region (V_(H) inFIG. 1) and at least three constant regions (CH₁, CH₂ and CH₃ in FIG.1). The variable regions determine the specificity of the antibody andthe constant regions have other functions.

Amino acid and structural information indicate that each variable regioncomprises three hypervariable regions (also known as complementaritydetermining regions or CDRs) flanked by four relatively conservedframework regions or FRs. The three CDRs, referred to as CDR₁, CDR₂, andCDR₃, are responsible for the binding specificity of individualantibodies. When antibodies are to be used as diagnostic and therapeuticagents, typically it is desirable to create antibodies that have thehighest binding specificity and affinity to the target molecule. It isbelieved that differences in the variable regions can have profoundeffects on the specificity and affinity of the antibody.

U.S. Pat. No. 5,707,624 describes the use of anti-HGF antibodies in thetreatment of Kaposi's sarcoma. Similarly, U.S. Pat. No. 5,997,868describes treating a tumor by administering an anti-HGF antibody to thepatient to be treated so as to block the ability of endogeneous HGF topromote angiogenesis in the tumor. More recently, investigators proposethat antibodies that bind the β-chain of HGF may have potential astherapeutic agents in patients with HGF-dependent tumors (Burgess (2006)supra).

Notwithstanding, there is still a need for additional HGF modulatorsthat can be used as therapeutic and diagnostic agents.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery of a family ofbinding proteins that specifically bind HGF, in particular, human HGF.The binding proteins are antibody-based in so far as they containantigen (i.e., HGF) binding sites based on the CDRs of a family ofantibodies that specifically bind HGF. The CDRs confer the bindingspecificity of the binding proteins to HGF. The binding proteins can beused as diagnostic and therapeutic agents. When used as a therapeuticagent, the binding proteins are engineered (e.g., humanized) so as toreduce or eliminate the risk of inducing an immune response against thebinding protein when administered to the recipient (e.g., a human).

The binding proteins neutralize the activity of HGF and, therefore, canbe used as a therapeutic agent. In certain embodiments, the bindingproteins prevent HGF from binding to its cognate receptor, c-Met,thereby neutralizing HGF activity. In other embodiments, the bindingproteins bind to HGF and neutralize its biological activity but withoutpreventing HGF from binding to the c-Met receptor. Because HGF has beenimplicated in the growth and proliferation of cancer cells, the bindingproteins can be used to inhibit the proliferation of cancer cells.Furthermore, when administered to a mammal, the binding proteins caninhibit or reduce tumor growth in the mammal.

These and other aspects and advantages of the invention will becomeapparent upon consideration of the following figures, detaileddescription, and claims.

DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1 is a schematic representation of a typical antibody.

FIG. 2 is a schematic diagram showing the amino acid sequence definingthe complete immunoglobulin heavy chain variable region of theantibodies denoted as 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6 and 3D11. Theamino acid sequences for each antibody are aligned against one anotherand the regions defining the signal peptide, CDR₁, CDR₂, and CDR₃ areidentified in boxes. The unboxed sequences represent FR sequences.

FIG. 3 is a schematic diagram showing the CDR₁, CDR₂, and CDR₃ sequencesfor each of the immunoglobulin heavy chain variable region sequencespresented in FIG. 2.

FIG. 4 is a schematic diagram showing the amino acid sequence definingthe complete immunoglobulin light chain variable region of theantibodies 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6, and 3D11. The amino acidsequences for each antibody are aligned against one another and theregions defining the signal peptide, CDR₁, CDR₂, and CDR₃ are identifiedin boxes. The unboxed sequences represent FR sequences.

FIG. 5 is a schematic diagram showing the CDR₁, CDR₂, and CDR₃ sequencesfor each of the immunoglobulin light chain variable region sequencespresented in FIG. 4.

FIG. 6 is a graph summarizing results from an experiment to measuretumor inhibitory activity of anti-HGF antibodies 1D3, 1F3, 1A3 and 2B8in a U87MG xenograft model. Diamonds correspond to PBS; trianglescorrespond to anti-HGF antibody 1A3; X corresponds to anti-HGF antibody1D3; squares correspond to anti-HGF antibody 1F3, and circles correspondto anti-HGF antibody 2B8.

FIG. 7 is a graph summarizing results from an experiment to measuretumor inhibitory activity of anti-HGF antibodies 1D3, 1F3, 1A3 and 2B8in a U118 xenograft model. Diamonds correspond to IgG; squarescorrespond to anti-HGF antibody 1F3, triangles to anti-HGF antibody 1D3;X corresponds to anti-HGF antibody 1A3; and circles correspond toanti-HGF antibody 2B8.

FIG. 8 is a table summarizing surface plasmon resonance data onantigen-binding affinity and kinetics of interaction between human HGFand chimeric, chimeric/humanized, or humanized 2B8 antibodies. The tablelists the pairs of Kappa light chain and IgG1 heavy chain tested. Thoseantibodies with standard deviations (STDEV) listed were analyzed inthree independent experiments.

FIG. 9 is a bar chart summarizing experimental data indicating thatHu2B8 binds an epitope mutually exclusive to murine monoclonal antibody2B8. Humanized or chimeric 2B8 was captured on an anti-human Fc chip.HGF then was bound to the humanized or chimeric 2B8. The ability ofmouse 2B8 or the control antibody (polyclonal goat anti-HGF antibody) tobind the captured HGF was measured. Both humanized 2B8 antibodies andchimeric 2B8 prevent murine 2B8 from binding HGF. White bars correspondto the chimeric 2B8 antibody; gray bars correspond to the humanizedHu2B8 antibody (kappa variable region Kv1-39.1 and heavy chain variableregion Hv5-51.1); black bars correspond to the humanized Hu2B8 antibody(kappa variable region Kv3-15.1 and heavy chain variable regionHv5-51.1).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, upon the discovery of a family ofbinding proteins that specifically bind, and neutralize the activity of,HGF, in particular, human HGF. The binding proteins can be used in avariety of diagnostic and therapeutic applications. The binding proteinsare based upon the antigen binding sites of certain monoclonalantibodies that have been selected for their ability to bind, andneutralize the activity of, HGF. In particular, the binding proteinscontain immunoglobulin variable region CDR sequences that togetherdefine a binding site for HGF.

In view of the neutralizing activity of these antibodies, they areparticularly useful in modulating the growth and/or proliferation of HGFresponsive cells, for example, cancer cells. When used as a therapeuticagent, the binding proteins can be engineered so as to minimize oreliminate the risk of inducing an immune response against the bindingproteins when administered to the recipient. Furthermore, depending uponthe particular application, it is contemplated that the binding proteinscan be conjugated to other moieties, for example, detectable labels, forexample, radiolabels, and effector molecules, for example, other proteinand small molecule-based therapeutics. Each of these features andaspects of the invention are discussed in more detail below.

I—Binding Proteins that Bind HGF

In one aspect, the invention provides an isolated binding protein thatbinds human HGF. The binding protein comprises (i) an immunoglobulinlight chain variable region comprising the structureCDR_(L1)-CDR_(L2)-CDR_(L3), and (ii) an immunoglobulin heavy chainvariable region comprising three complementarity determining regions(CDRs), wherein the immunoglobulin light chain variable region and theimmunoglobulin heavy chain variable region together define a singlebinding site for binding human HGF. CDR_(L1) comprises the amino acidsequence X₁ X₂ Ser X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅, whereinamino acid X₁ is Arg, Lys, or Ser, X₂ is Ala or Thr, X₄ is Glu, Gln, orSer, X₅ is Asn, Asp, or Ser, X₆ is Ile or Val, X₇ is Asp, Lys, Ser, Val,or Tyr, X₈ is a peptide bond or Tyr, X₉ is a peptide bond or Asp, X₁₀ isa peptide bond or Gly, X₁₁ is a peptide bond or Asn, X₁₂ is a peptidebond, Ile, or Ser, X₁₃ is Asn or Tyr, X₁₄ is Ile, Leu, Met, or Val, X₁₅is Ala, Asn, His, or Ser. CDR_(L2) comprises the amino acid sequence X₁₆X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂, wherein amino acid X₁₆ is Ala, Asp, Arg, Gly,or Val, X₁₇ is Ala, Thr, or Val, X₁₈ is Asn, Ser, or Thr, X₁₉ is Arg,Asn, Lys, or His, X₂₀ is Leu or Arg, X₂₁ is Ala, Asn, Glu, Val, or Pro,X₂₂ is Asp, Ser, or Thr. CDR_(L3) comprises the amino acid sequence X₂₃X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ Pro X₃₀ Thr, wherein amino acid X₂₃ is Leu, Gly, orGln, X₂₄ is His or Gln, X₂₅ is Phe, Ser, Trp, or Tyr, X₂₆ is Asp, Ile,Ser, Trp, or Tyr, X₂₇ is Gly, Glu, Asn, or Ser, X₂₈ is Asp, Asn, Phe,Thr, or Tyr, X₃₀ is Leu, Phe, Pro, or Tyr.

In another aspect, the invention provides an isolated binding proteinthat binds human HGF comprising (i) an immunoglobulin heavy chainvariable region comprising the structure CDR_(H1)-CDR_(H2)-CDR_(H3) and(ii) an immunoglobulin light chain variable region comprising threecomplementarity determining regions (CDRs), wherein the immunoglobulinheavy chain variable region and the immunoglobulin light chain variableregion together define a single binding site for binding human HGF.CDR_(H1) comprises the amino acid sequence X₁ Tyr X₃ X₄ X₅, whereinamino acid X₁ is Asp, Asn, Ser, or Thr, X₃ is Phe, Ser, Trp, or Tyr, X₄is Ile, Leu, or Met, X₅ is Asn, His, or Ser. CDR_(H2) comprises theamino acid sequence X₆ Ile X₈ X₉ X₁₀ X₁₁ Gly X₁₃ X₁₄ X₁₅ Tyr X₁₇ X₁₈ X₁₉X₂₀ X₂₁ X₂₂, wherein amino acid X₆ is Lys, Gln, Glu, Val, or Tyr, X₈ isAsn, Gly, Ser, Trp, or Tyr, X₉ is Ala, Pro or Ser, X₁₀ is Gly or Thr,X₁₁ is a peptide bond, Asp, Asn, Gly, or Ser, X₁₃ is Asp, Asn, His, orSer, X₁₄ is Ser or Thr, X₁₅ is Asn or Tyr, X₁₇ is Asn or Pro, X₁₈ isAla, Asp, Gly, Gln, Glu, Pro, or Ser, X₁₉ is Asn, Lys, Met, or Ser, X₂₀is Leu, Phe or Val, X₂₁ is Lys, Met, or Gln, X₂₂ is Asp, Gly or Ser.CDR_(H3) comprises the amino acid sequence X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉X₃₀ X₃₁ X₃₂ X₃₃ X₃₄ Tyr, wherein amino acid X₂₃ is Arg, Asn, Gln, orGlu, X₂₄ is Gly, Leu, Arg, or Tyr, X₂₅ is a peptide bond, Asp, or Gly,X₂₆ is a peptide bond or Gly, X₂₇ is a peptide bond or Tyr, X₂₈ is apeptide bond, Leu, or Tyr, X₂₉ is a peptide bond, Gly, Leu, Arg, or Val,X₃₀ is a peptide bond, Asp, Gly, or Glu, X₃₁ is a peptide bond, Asn,Arg, Ser, or Tyr, X₃₂ is peptide bond, Ala, Gly, Ile, or Tyr, X₃₃ is Metor Phe, X₃₄ is Ala or Asp.

It is understood that the binding protein can comprise both theimmunoglobulin light chain and the immunoglobulin heavy chain sequencesor the fragments thereof, noted above. Furthermore, it is understoodthat the binding protein can be an intact antibody or an antigen bindingfragment thereof, or a biosynthetic antibody site.

In certain embodiments, the CDR sequences of the immunoglobulin lightchain and the immunoglobulin heavy chain are interposed with frameworkregions (FR).

In certain other embodiments, the CDR sequences of the immunoglobulinlight chain and the immunoglobulin heavy chain are interposed betweenhuman or humanized framework regions.

In another aspect, the invention provides an isolated binding proteinthat specifically binds human HGF. The binding protein comprises: (a) animmunoglobulin light chain variable region comprising the structureCDR_(L1)-CDR_(L2)-CDR_(L3) and (b) immunoglobulin heavy chain variableregion, wherein the immunoglobulin light chain variable region and theimmunoglobulin heavy chain variable region together define a singlebinding site for binding human HGF. The CDR_(L1) comprises a sequenceselected from the group consisting of SEQ ID NO. 8 (1A3), SEQ ID NO. 18(2B8), SEQ ID NO. 28 (2F8), SEQ ID NO. 38 (3B6), SEQ ID NO. 48 (3D11),SEQ ID NO. 58 (1D3), SEQ ID NO. 68 (1F3), and SEQ ID NO. 78 (3A12). TheCDR_(L2) comprises a sequence selected from the group consisting of SEQID NO. 9 (1A3), SEQ ID NO. 19 (2B8), SEQ ID NO. 29 (2F8), SEQ ID NO. 39(3B6), SEQ ID NO. 49 (3D11), SEQ ID NO. 59 (1D3), SEQ ID NO. 69 (1F3),SEQ ID NO. 79 (3A12) and SEQ ID NO. 206 (LRMR2B8LC). The CDR_(L3)comprises a sequence selected from the group consisting of SEQ ID NO. 10(1A3), SEQ ID NO. 20 (2B8), SEQ ID NO. 30 (2F8), SEQ ID NO. 40 (3B6),SEQ ID NO. 50 (3D11), SEQ ID NO. 60 (1D3), SEQ ID NO. 70 (1F3), and SEQID NO. 80 (3A12). Throughout the specification and claims, the sequencesdenoted by a particular SEQ ID NO. are followed in parentheses by theantibody that was the origin of the particular sequence. By way ofexample, SEQ ID NO. 8 (1A3) indicates that the sequence of SEQ ID NO. 8is based upon the sequence present in antibody 1A3.

In one embodiment, the binding protein comprises an immunoglobulin lightchain variable region comprising a CDR_(L1) comprising the sequence ofSEQ ID NO. 8 (1A3), a CDR_(L2) comprising the sequence of SEQ ID NO. 9(1A3), and a CDR_(L3) comprising the sequence of SEQ ID NO. 10 (1A3).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 18 (2B8), a CDR_(L2) comprising the sequence ofSEQ ID NO. 19 (2B8) or SEQ ID NO. 206 (LRMR2B8LC), and a CDR_(L3)comprising the sequence of SEQ ID NO. 20 (2B8).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 28 (2F8), a CDR_(L2) comprising the sequence ofSEQ ID NO. 29 (2F8), and a CDR_(L3) comprising the sequence of SEQ IDNO. 30 (2F8).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 38 (3B6), a CDR_(L2) comprising the sequence ofSEQ ID NO. 39 (3B6), and a CDR_(L3) comprising the sequence of SEQ IDNO. 40 (3B6).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 48 (3D11), a CDR_(L2) comprising the sequence ofSEQ ID NO. 49 (3D11), and a CDR_(L3) comprising the sequence of SEQ IDNO. 50 (3D11).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 58 (1D3), a CDR_(L2) comprising the sequence ofSEQ ID NO. 59 (1D3), and a CDR_(L3) comprising the sequence of SEQ IDNO. 60 (1D3).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 68 (1F3), a CDR_(L2) comprising the sequence ofSEQ ID NO. 69 (1F3), and a CDR_(L3) comprising the sequence of SEQ IDNO. 70 (1F3).

In another embodiment, the binding protein comprises an immunoglobulinlight chain variable region comprising a CDR_(L1) comprising thesequence of SEQ ID NO. 78 (3A12), a CDR_(L2) comprising the sequence ofSEQ ID NO. 79 (3A12), and a CDR_(L3) comprising the sequence of SEQ IDNO. 80 (3A12).

In each of the foregoing embodiments, the CDR_(L1), CDR_(L2), andCDR_(L3) sequences preferably are interposed between human or humanizedimmunoglobulin FRs. It is understood that the binding protein can be anintact antibody, an antigen binding fragment thereof, or a biosyntheticantibody site.

In another aspect, the invention provides an isolated binding proteinthat binds human HGF. The binding protein comprises (a) animmunoglobulin heavy chain variable region comprising the structureCDR_(H1)-CDR_(H2)-CDR_(H3), and (b) an immunoglobulin light chainvariable region, wherein the immunoglobulin heavy chain variable regionand the immunoglobulin light chain variable region together define asingle binding site for binding human HGF. The CDR_(H1) comprises asequence selected from the group consisting of SEQ ID NO. 5 (1A3), SEQID NO. 15 (2B8), SEQ ID NO. 25 (2F8), SEQ ID NO. 35 (3B6), SEQ ID NO. 45(3D11), SEQ ID NO. 55 (1D3), SEQ ID NO. 65 (1F3), and SEQ ID NO. 75(3A12); the CDR_(H2) comprises a sequence selected from the groupconsisting of SEQ ID NO. 6 (1A3), SEQ ID NO. 16 (2B8), SEQ ID NO. 26(2F8), SEQ ID NO. 36 (3B6), SEQ ID NO. 46 (3D11), SEQ ID NO. 56 (1D3),SEQ ID NO. 66 (1F3), SEQ ID NO. 76 (3A12), SEQ ID NO. 202 (Hu2B8Hv1f.1), SEQ ID NO. 203 (Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1), SEQ ID NO. 204(LR2B8HC) and SEQ ID NO. 205 (LRMR2B8HC); and the CDR_(H3) comprises asequence selected from the group consisting of SEQ ID NO. 7 (1A3), SEQID NO. 17 (2B8), SEQ ID NO. 27 (2F8), SEQ ID NO. 37 (3B6), SEQ ID NO. 47(3D11), SEQ ID NO. 57 (1D3), SEQ ID NO. 67 (1F3), and SEQ ID NO. 77(3A12).

In one embodiment, the binding protein comprises an immunoglobulin heavychain variable region comprising: a CDR_(H1) comprising the sequence ofSEQ ID NO. 5 (1A3); a CDR_(H2) comprising the sequence of SEQ ID NO. 6(1A3); and a CDR_(H3) comprising the sequence of SEQ ID NO. 7 (1A3).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising: a CDR_(H1) comprising thesequence of SEQ ID NO. 15 (2B8); a CDR_(H2) comprising the sequence ofSEQ ID NO. 16 (2B8), SEQ ID NO. 202 (Hu2B8 Hv1f.1), SEQ ID NO. 203(Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1), SEQ ID NO. 204 (LR2B8HC) or SEQ ID NO.205 (LRMR2B8HC); and a CDR_(H3) comprising the sequence of SEQ ID NO. 17(2B8).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising: a CDR_(H1) comprising thesequence of SEQ ID NO. 25 (2F8); a CDR_(H2) comprising the sequence ofSEQ ID NO. 26 (2F8); and a CDR_(H3) comprising the sequence of SEQ IDNO. 27 (2F8).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising a CDR_(H1) comprising thesequence of SEQ ID NO. 35 (3B6); a CDR_(H2) comprising the sequence ofSEQ ID NO. 36 (3B6); and a CDR_(H3) comprising the sequence of SEQ IDNO. 37 (3B6).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising: a CDR_(H1) comprising thesequence of SEQ ID NO. 45 (3D11); a CDR_(H2) comprising the sequence ofSEQ ID NO. 46 (3D11); and a CDR_(H3) comprising the sequence of SEQ IDNO. 47 (3D11).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising: a CDR_(H1) comprising thesequence of SEQ ID NO. 55 (1D3); a CDR_(H2) comprising the sequence ofSEQ ID NO. 56 (1D3); and a CDR_(H3) comprising the sequence of SEQ IDNO. 57 (1D3).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising: a CDR_(H1) comprising thesequence of SEQ ID NO. 65 (1F3); a CDR_(H2) comprising the sequence ofSEQ ID NO. 66 (1F3); and a CDR_(H3) comprising the sequence of SEQ IDNO. 67 (1F3).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising: a CDR_(H1) comprising thesequence of SEQ ID NO. 75 (3A12); a CDR_(H2) comprising the sequence ofSEQ ID NO. 76 (3A12); and a CDR_(H3) comprising the sequence of SEQ IDNO. 77 (3A12).

In each of the foregoing embodiments, the CDR_(H1), CDR_(H2), andCDR_(H3) sequences preferably are interposed between human or humanizedimmunoglobulin FRs. It is understood that the binding protein can be anintact antibody, an antigen binding fragment thereof, or a biosyntheticantibody site.

In another aspect, the invention provides a binding protein that bindshuman HGF. The binding protein comprises an immunoglobulin heavy chainvariable region selected from the group consisting of residues 20-141 ofSEQ ID NO. 2 (1A3), residues 20-137 of SEQ ID NO. 12 (2B8), residues20-137 of SEQ ID NO. 22 (2F8), residues 20-139 of SEQ ID NO. 32 (3B6),residues 20-132 of SEQ ID NO. 42 (3D11), residues 20-141 of SEQ ID NO.52 (1D3), residues 20-141 of SEQ ID NO. 62 (1F3), and residues 20-141 ofSEQ ID NO. 72 (3A12) and an immunoglobulin light chain variable regionselected from the group consisting of residues 21-127 of SEQ ID NO. 4(1A3), residues 21-127 of SEQ ID NO. 14 (2B8), residues 20-131 of SEQ IDNO. 24 (2F8), residues 23-129 of SEQ ID NO. 34 (3B6), residues 23-128 ofSEQ ID NO. 44 (3D11), residues 21-127 of SEQ ID NO. 54 (1D3), residues21-127 of SEQ ID NO. 64 (1F3), and residues 21-127 of SEQ ID NO. 74(3A12).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-141 of SEQ ID NO. 2 (1A3), and an immunoglobulin light chainvariable region comprising the amino acid sequence of residues 21-127 ofSEQ ID NO. 4 (1A3).

In one embodiment, the binding protein comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of residues20-137 of SEQ ID NO. 12 (2B8), and an immunoglobulin light chainvariable region comprising the amino acid sequence of residues 21-127 ofSEQ ID NO. 14 (2B8).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-137 of SEQ ID NO. 22 (2F8), and an immunoglobulin lightchain variable region comprising the amino acid sequence of residues20-131 of SEQ ID NO. 24 (2F8).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-139 of SEQ ID NO. 32 (3B6), and an immunoglobulin lightchain variable region comprising the amino acid sequence of residues23-129 of SEQ ID NO. 34 (3B6).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-132 of SEQ ID NO. 42 (3D11), and an immunoglobulin lightchain variable region comprising the amino acid sequence of residues23-128 of SEQ ID NO. 44 (3D11).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-141 of SEQ ID NO. 52 (1D3), and an immunoglobulin lightchain variable region comprising the amino acid sequence of residues21-127 of SEQ ID NO. 54 (1D3).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-141 of SEQ ID NO. 62 (1F3), and an immunoglobulin lightchain variable region comprising the amino acid sequence of residues21-127 of SEQ ID NO. 64 (1F3).

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising the amino acid sequence ofresidues 20-141 of SEQ ID NO. 72 (3A12), and an immunoglobulin lightchain variable region comprising the amino acid sequence of residues21-127 of SEQ ID NO. 74 (3A12).

In each of the foregoing embodiments, the binding protein can be anintact antibody, an antigen binding fragment thereof, or a biosyntheticantibody site.

In another aspect, the invention provides an isolated binding proteinthat binds human HGF. The binding protein comprises (i) animmunoglobulin light chain variable region selected from the groupconsisting of SEQ ID NO. 173 (Hu2B8 Kv1-39.1 light chain variableregion), SEQ ID NO. 179 (Hu2B8 Kv3-15.1 light chain variable region),SEQ ID NO. 193 (LR2B8LC light chain variable region), and SEQ ID NO. 199(LRMR2B8LC light chain variable region); and (ii) an immunoglobulinheavy chain variable region selected from the group consisting of SEQ IDNO. 159 (Hu2B8 Hv1f.1 heavy chain variable region), SEQ ID NO. 165(Hu2B8 Hv5a.1 heavy chain variable region), SEQ ID NO. 169 (Hu2B8Hv5-51.1 heavy chain variable region), SEQ ID NO. 183 (LR2B8HC heavychain variable region), and SEQ ID NO. 189 (LRMR2B8LC light chainvariable region). The binding protein can be an intact antibody, anantigen binding fragment thereof, or a biosynthetic antibody site.

In another aspect, the invention provides an isolated binding proteinthat binds human HGF. The binding protein comprises (i) animmunoglobulin light chain selected from the group consisting of SEQ IDNO. 177 (Hu2B8 Kv1-39.1+kappa constant (Km(3) allotype (allele 2)), SEQID NO. 181 (Hu2B8 Kv3-15.1+Kappa constant (Km(3) allotype (allele 2)),SEQ ID NO. 197 (LR2B8LC+Kappa constant (Km(3) allotype (allele 1)), andSEQ ID NO. 201 (LRMR2B8LC+Kappa constant (Km(3) allotype (allele 1));and (ii) an immunoglobulin heavy chain selected from the groupconsisting of SEQ ID NO. 163 (Hu2B8 Hv1f.1+IgG1 Constant (G1m(17,1)allotype)), SEQ ID NO. 167 (Hu2B8 Hv5a.1+IgG1 Constant (G1m(17,1)allotype)), SEQ ID NO. 171 (Hu2B8 Hv5-51.1+IgG1 Constant (G1m(17,1)allotype)), SEQ ID NO. 187 (LR2B8HC+IgG1 Constant (G1m(3) allotype)(allele 1)), and SEQ ID NO. 191 (LRMR2B8HC+IgG1 Constant (G1m(3)allotype) (allele 1)). The binding protein can be an intact antibody, anantigen binding fragment thereof, or a biosynthetic antibody site.

In another aspect, the invention provides an isolated binding proteinthat binds reduced human HGF. The binding protein comprises (i) animmunoglobulin light chain variable region comprising three CDRs, and(ii) an immunoglobulin heavy chain variable region comprising threeCDRs. The CDRs typically are interposed between FRs. The CDRs of theimmunoglobulin light chain and the immunoglobulin heavy chain togetherdefine a binding site that binds reduced human HGF, for example, theα-chain of reduced HGF. Reduced HGF refers to HGF treated with an amountof reducing agent, for example, dithiothreitol (DTT), 2-mercaptoethanol,or glutathione sufficient to reduce the disulfide linkage between theα-chain and the β-chain. Exemplary concentrations include, for example,100 mM DTT and 5% 2-mercaptoethanol.

In certain embodiments, the binding protein comprises an immunoglobulinlight chain variable region comprising at least one CDR selected fromthe group consisting of CDR_(L1), CDR_(L2) and CDR_(L3). Optionally, thebinding protein comprises two CDRs, for example, CDR_(L1) and CDR_(L2),or CDR_(L1) and CDR_(L3), or CDR_(L1) and CDR_(L3). Optionally, thebinding protein comprises all three CDRs, i.e., CDR_(L1), CDR_(L2) andCDR_(L3). CDR_(L1) comprises the amino acid sequence X₁ X₂ Ser X₄ X₅ X₆X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅, wherein amino acid X₁ is Arg or Lys,X₂ is Ala or Thr, X₄ is Glu or Gln, X₅ is Asn, Ser, or Asp, X₆ is Ile orVal, X₇ is Tyr, Asp, or Lys, X₈ is a peptide bond or Tyr, X₉ is apeptide bond or Asp, X₁₀ is a peptide bond or Gly, X₁₁ is a peptide bondor Asn, X₁₂ is a peptide bond or Ser, X₁₃ is Asn or Tyr, X₁₄ is Ile orLeu, X₁₅ is Ala, Asn, or Ser. CDR_(L2) comprises the amino acid sequenceX₁₆ X₁₇ X₁₈ X₁₉ Leu X₂₁ X₂₂, wherein amino acid X₁₆ is Ala, Asp, Val, orArg, X₁₇ is Ala or Val, X₁₈ is Asn, Ser, or Thr, X₁₉ is Arg, Asn, orHis, X₂₁ is Ala, Glu, Val, or Pro, X₂₂ is Asp or Ser. CDR_(L3) comprisesthe amino acid sequence X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ Pro X₃₀ Thr, whereinamino acid X₂₃ is Leu or Gln, X₂₄ is His or Gln, X₂₅ is Phe, Ser, orTyr, X₂₆ is Asp, Ile, or Trp, X₂₇ is Gly or Glu, X₂₈ is Asp, Phe, orThr, X₃₀ is Phe, Pro, or Tyr.

In another embodiment, the binding protein comprises an immunoglobulinheavy chain variable region comprising at least one CDR selected fromthe group consisting of CDR_(H1), CDR_(H2), and CDR_(H3). Optionally,the binding protein comprises two CDRs, for example, CDR_(H1) andCDR_(H2), or CDR_(H1) and CDR_(H3), or CDR_(H1) and CDR_(H3).Optionally, the binding protein comprises all three CDRs, i.e.,CDR_(H1), CDR_(H2) and CDR_(H3). CDR_(H1) comprises the amino acidsequence X₁ Tyr X₃ X₄ X₅, wherein amino acid X₁ is Asp, Asn, Ser, orThr, X₃ is Phe, Trp, or Tyr, X₄ is Ile or Met, X₅ is Asn, His, or Ser.CDR_(H2) comprises the amino acid sequence X₆ Ile X₈ X₉ Gly X₁₁ Gly X₁₃X₁₄ X₁₅ Tyr X₁₇ X₁₈ X₁₉ X₂₀ Lys X₂₂, wherein amino acid X₆ is Lys, Gln,or Tyr, X₈ is Gly, Ser, or Tyr, X₉ is Pro or Ser, X₁₁ is Asp, Gly, orSer, X₁₃ is Asp or Ser, X₁₄ is Ser or Thr, X₁₅ is Asn or Tyr, X₁₇ is Asnor Pro, X₁₈ is Ala, Asp, Gly, or Glu, X₁₉ is Asn, Met, or Ser, X₂₀ isPhe or Val, X₂₂ is Asp or Gly. CDR_(H3) comprises the amino acidsequence X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ X₃₁ X₃₂ X₃₃ Asp Tyr, whereinamino acid X₂₃ is Arg or Gln, X₂₄ is Gly or Leu, X₂₅ is Asp, Gly, or apeptide bond, X₂₆ is Gly or a peptide bond, X₂₇ is a peptide bond orTyr, X₂₈ is Leu, a peptide bond or Tyr, X₂₉ is a Gly, Arg or Leu, X₃₀ isAsp, Gly or Glu, X₃₁ is a Tyr, Arg or Asn, X₃₂ is Ala, Gly or Tyr, X₃₃is Met or Phe.

It is understood that the binding protein can comprise both theimmunoglobulin heavy chain and the immunoglobulin light chain sequencesor the fragments thereof, noted above. Furthermore, it is understoodthat the binding protein can be an intact antibody or an antigen bindingfragment thereof, or a biosynthetic antibody site.

In certain embodiments, the binding protein comprises an immunoglobulinlight chain variable region comprising (i) a CDR_(L1) having a sequenceselected from the group consisting of SEQ ID NO. 8 (1A3), SEQ ID NO. 28(2F8), SEQ ID NO. 38 (3B6), SEQ ID NO. 58 (1D3), and SEQ ID NO. 68(1F3), (ii) a CDR_(L2) having a sequence selected from the groupconsisting of SEQ ID NO. 9 (1A3), SEQ ID NO. 29 (2F8), SEQ ID NO. 39(3B6), SEQ ID NO. 59 (1D3), and SEQ ID NO. 69 (1F3), and (iii) aCDR_(L3) having a sequence selected from the group consisting of SEQ IDNO. 10 (1A3), SEQ ID NO. 30 (2F8), SEQ ID NO. 40 (3B6), SEQ ID NO. 60(1D3), and SEQ ID NO. 70 (1F3). The CDR sequences can be interposedbetween human or humanized FRs. In other embodiments, the bindingprotein comprises an immunoglobulin light chain variable regioncomprising an amino acid sequence selected from the group consisting ofresidues 21-127 of SEQ ID NO. 4 (1A3), residues 20-131 of SEQ ID NO. 24(2F8), residues 23-129 of SEQ ID NO. 34 (3B6), residues 21-127 of SEQ IDNO. 54 (1D3), and residues 21-127 of SEQ ID NO. 64 (1F3).

In certain other embodiments, the binding protein comprises animmunoglobulin heavy chain variable region comprising (i) a CDR_(H1)having a sequence selected from the group consisting of SEQ ID NO. 5(1A3), SEQ ID NO. 25 (2F8), SEQ ID NO. 35 (3B6), SEQ ID NO. 55 (1D3),and SEQ ID NO. 65 (1F3), (ii) a CDR_(H2) having a sequence selected fromthe group consisting of SEQ ID NO. 6 (1A3), SEQ ID NO. 26 (2F8), SEQ IDNO. 36 (3B6), SEQ ID NO. 56 (1D3), and SEQ ID NO. 66 (1F3), and (iii) aCDR_(H3) having a sequence selected from the group consisting of SEQ IDNO. 7 (1A3), SEQ ID NO. 27 (2F8), SEQ ID NO. 37 (3B6), SEQ ID NO. 57(1D3), and SEQ ID NO. 67 (1F3). The CDR sequences can be interposedbetween human or humanized FRs. In another embodiment, theimmunoglobulin heavy chain variable region comprises an amino acidsequence selected from the group consisting of residues 20-141 of SEQ IDNO. 2 (1A3), residues 20-137 of SEQ ID NO. 22 (2F8), residues 20-139 ofSEQ ID NO. 32 (3B6), residues 20-141 of SEQ ID NO. 52 (1D3), andresidues 20-141 of SEQ ID NO. 62 (1F3).

In another aspect, the invention provides an isolated binding proteinthat binds human HGF and comprises an immunoglobulin light chainvariable region and an immunoglobulin heavy chain variable region. Theisolated binding protein competes for binding to HGF with at least onereference antibody selected from the group consisting of (i) an antibodyhaving an immunoglobulin light chain variable region of residues 20-131of SEQ ID NO. 24 (2F8), and an immunoglobulin heavy chain variableregion of residues 20-137 of SEQ ID NO. 22 (2F8), (ii) an antibodyhaving an immunoglobulin light chain variable region of residues 23-129of SEQ ID NO. 34 (3B6), and an immunoglobulin heavy chain variableregion of residues 20-139 of SEQ ID NO. 32 (3B6), and (iii) an antibodyhaving an immunoglobulin light chain variable region of residues 23-128of SEQ ID NO. 44 (3D11), and an immunoglobulin heavy chain variableregion of residues 20-132 of SEQ ID NO. 42 (3D11). Under certaincircumstances, the binding protein binds the same epitope of HGF as oneof the reference antibodies.

It is understood that each of the binding proteins discussed above canbe an intact antibody, for example, a monoclonal antibody.Alternatively, the binding protein can be an antigen binding fragment ofan antibody, or can be a biosynthetic antibody binding site. Antibodyfragments include Fab, Fab′, (Fab′)₂ or Fv fragments. Techniques formaking such antibody fragments are known to those skilled in the art. Anumber of biosynthetic antibody binding sites are known in the art andinclude, for example, single Fv or sFv molecules, described, forexample, in U.S. Pat. No. 5,476,786. Other biosynthetic antibody bindingsites include bispecific or bifunctional binding proteins, for example,bispecific or bifunctional antibodies, which are antibodies or antibodyfragments that bind at least two different antigens. For example,bispecific binding proteins can bind HGF, for example, human HGF, andanother antigen of interest. Methods for making bispecific antibodiesare known in art and, include, for example, by fusing hybridomas or bylinking Fab′ fragments. See, e.g., Songsivilai et al. (1990) CLIN. EXP.IMMUNOL. 79: 315-325; Kostelny et al. (1992) J. IMMUNOL. 148: 1547-1553.

The binding proteins of the invention can bind hHGF containing acysteine to arginine substitution at position 561 or a glycine toglutamate substitution at position 555.

In another aspect, the invention provides an isolated binding proteinthat binds human HGF with a k_(d) of 4.0×10⁻⁵ s⁻¹ or lower, 3.0×10⁻⁵ s⁻¹or lower, or 2.0×10⁻⁵ s⁻¹ or lower. The isolated binding proteins canbind human HGF with a k_(d) from 5.0×10⁻⁵ s⁻¹ to 0.5×10⁻⁵ s⁻¹, or from4.0×10⁻⁵ s⁻¹ to 1.0×10⁻⁵ s⁻¹, or from 3.0×10⁻⁵ s⁻¹ to 1.5×10⁻⁵ s⁻¹. Inanother aspect, the invention provides an isolated binding protein thatbinds human HGF with a K_(D) of 100 pM or lower, or 20 pM or lower, or10 pM or lower, or 5 pM or lower. The isolated binding proteins can bindhuman HGF with a K_(D) from 100 pM to 5 pM, or from 20 pM to 5 pM, orfrom 15 pM to 10 pM, or from 20 pM to 10 pM, or from 15 pM to 5 pM.Unless otherwise specified, K_(D) values are determined by the methods,and under the conditions, described in Example 6.

In another aspect, the invention provides an isolated binding proteinthat binds human HGF, wherein the antibody binds to human HGF with lowerK_(D) at 37° C. than at 25° C. The binding protein binding optionallybinds human HGF with a K_(D) less than 5 pM at 37° C.

In other aspects and embodiments, the binding proteins can inhibit hHGFfrom binding to c-Met. For example, the binding proteins can have anIC₅₀ (concentration at 50% of maximum inhibition) of at least about 4.0,4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 nM when assayed using the protocoldescribed in Example 7(a). In certain other embodiments, the bindingproteins can neutralize HGF BrdU incorporation in 4 MBr-5 cells (ATCC,Catalog No. CCL208) using the method described in Example 7(b).

The binding proteins have an IC₅₀ of 50 nM or lower, preferably 45, 40,35, 30, 25, 20, 15, 10, 5, 1, 0.5 nM or lower, when assayed using theprotocol described in Example 7(b). In certain other embodiments, thebinding proteins can be used to inhibit HGF stimulated c-Metphosphorylation in PC-3 cells (ATCC, Manassus, Va. Catalog No. CRL-1435)using the assay described in Example 9. The binding proteins inhibitHGF-stimulated (1.25 nM) c-Met phosphorylation in PC-3 cells with anIC₅₀ of 2 nM or less (Table 8), using the assay described in Example 9.

II—Production of Binding Proteins

Binding proteins of the invention can be produced in various ways usingapproaches know in the art. For example, DNA molecules encoding lightchain variable regions and heavy chain variable regions can bechemically synthesized, using a commercial synthesizer and sequenceinformation provided herein. Such synthetic DNA molecules can be ligatedto other appropriate nucleotide sequences, including, e.g., constantregion coding sequences, and expression control sequences, to produceconventional gene expression constructs encoding the desired bindingproteins. Production of defined gene constructs is within routine skillin the art. Alternatively, the sequences provided herein can be clonedout of hybridomas by conventional hybridization techniques or PCRtechniques, using synthetic nucleic acid probes whose sequences arebased on sequence information provided herein or prior art sequenceinformation regarding genes encoding the heavy and light chains ofmurine antibodies in hybridoma cells. Production and use of such probesis within ordinary skill in the art.

The nucleic acids encoding the desired binding proteins can beintroduced (ligated) into expression vectors, which can be introducedinto a host cell via standard transfection or transformation techniquesknown in the art. Exemplary host cells include, for example, E. colicells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney (BHK) cells, monkey kidney cells (COS), human hepatocellularcarcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwiseproduce immunoglobulin protein. Transfected host cells can be grownunder conditions that permit the host cells to express the genes ofinterest, for example, the genes that encode the immunoglobulin light orheavy chain variable regions. The resulting expression products can beharvested using techniques known in the art.

The particular expression and purification conditions will varydepending upon what expression system is employed. For example, if thegene is to be expressed in E. coli, it is first cloned into anexpression vector. This is accomplished by positioning the engineeredgene downstream from a suitable bacterial promoter, e.g., Trp or Tac,and a signal sequence, e.g., a sequence encoding fragment B of protein A(FB). The resulting expressed fusion protein typically accumulates inrefractile or inclusion bodies in the cytoplasm of the cells, and may beharvested after disruption of the cells by French press or sonication.The refractile bodies then are solubilized, and the expressed proteinsrefolded and cleaved by the methods already established for many otherrecombinant proteins.

If the engineered gene is to be expressed in eukayotic host cells, forexample, myeloma cells or CHO cells, it is first inserted into anexpression vector containing a suitable eukaryotic promoter, a secretionsignal, immunoglobulin enhancers, and various introns. This expressionvector optionally can contain sequences encoding all or part of aconstant region, enabling an entire, or a part of, a heavy or lightchain to be expressed. The gene construct can be transfected intomyeloma cells or CHO cells using established transfection protocols.Such transfected cells can express V_(L) or V_(H) fragments, V_(L)-V_(H)heterodimers, V_(H)-V_(L) or V_(L)-V_(H) single chain polypeptides,complete heavy or light immunoglobulin chains, or portions thereof, eachof which may be attached to a protein domain having another function(e.g., cytotoxicity).

III—Modifications to the Binding Proteins

It is understood that the binding proteins can be modified to optimizeperformance depending upon the intended use of the binding proteins. Forexample, when the binding protein is being used as a therapeutic agent,the binding protein can be modified to reduce its immunogenicity in theintended recipient. Alternatively or in addition, the binding proteincan be fused or coupled to another protein or peptide, for example, agrowth factor, cytokine, or cytotoxin. Such modifications can beachieved by using routine gene manipulation techniques known in the art.

Various techniques for reducing the antigenicity of antibodies andantibody fragments are known in the art. These techniques can be used toreduce or eliminate the antigenicity of the binding proteins of theinvention. For example, when the binding proteins are to be administeredto a human, the binding proteins preferably are engineered to reducetheir antigenicity in humans. This process often is referred to ashumanization. Preferably, the humanized binding proteins have the sameor substantially the same affinity for the antigen as the originalnon-humanized binding protein it was derived from.

In one well known humanization approach, chimeric proteins are createdin which immunoglobulin constant regions of antibodies from one species,e.g., mouse, are replaced with immunoglobulin constant regions from asecond, different species, e.g., a human. In this example, the resultingantibody is a mouse-human chimera, where the human constant regionsequences, in principle, are less immunogenic than the counterpartmurine sequences. This type of antibody engineering is described, forexample, Morrison, et al. (1984) PROC. NAT. ACAD. SCI. 81: 6851-6855,Neuberger et al. (1984) NATURE 312: 604-608; U.S. Pat. Nos. 6,893,625(Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).

In another approach, known as CDR grafting, the CDRs of the light andheavy chain variable regions of an antibody of interest are grafted intoframeworks (FRs) from another species. For example, murine CDRs can begrafted into human FR sequences. In some embodiments, the CDRs of thelight and heavy chain variable regions of an anti-HGF antibody aregrafted into human FRs or consensus human FRs. In order to createconsensus human FRs, FRs from several human heavy chain or light chainamino acid sequences are aligned to identify a consensus amino acidsequence. CDR grafting is described, for example, in U.S. Pat. Nos.7,022,500 (Queen); 6,982,321 (Winter); 6,180,370 (Queen); 6,054,297(Carter); 5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen);5,565,332 (Hoogenboom); 5,585,089 (Queen); 5,530,101 (Queen); Jones etal. (1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332:323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter(1998) FEBS LETT 430: 92-94.

In an approach called “superhumanization,” antibodies in which humanimmunogenicity is reduced or eliminated are created by an alternativeform of grafting. In superhumanization, human FR sequences are chosenfrom a set of human germline genes based on the structural similarity ofthe human CDRs to those of the mouse antibody to be humanized. Thisapproach is described, for example, in U.S. Pat. No. 6,881,557 (Foote)and in Tan et al. (2002) J. IMMUNOL 169:1119-1125.

Other approaches to reduce immunogenicity include, techniques are knownas “reshaping,” “hyperchimerization,” or “veneering/resurfacing” toproduce humanized antibodies. See, e.g., Vaswami et al. (1998) ANNALS OFALLERGY, ASTHMA, & IMMUNOL. 81: 105; Roguska et al. (1996) PROT.ENGINEER 9: 895-904; and U.S. Pat. No. 6,072,035 (Hardman). In theveneering/resurfacing approach, the surface accessible amino acidresidues in the murine antibody are replaced by amino acid residues morefrequently found at the same positions in a human antibody. This type ofantibody resurfacing is described, for example, in U.S. Pat. No.5,639,641 (Pedersen).

One exemplary approach for converting a mouse antibody into a formsuitable for medical use in humans is known as ACTIVMAB™ technology(Vaccinex, Inc., Rochester, N.Y.), which involves a vaccinia virus-basedvector to express antibodies in mammalian cells. High levels ofcombinatorial diversity of immunoglobulin heavy and light chains aresaid to be produced. See, e.g., U.S. Pat. Nos. 6,706,477 (Zauderer);6,800,442 (Zauderer); and 6,872,518 (Zauderer).

Another exemplary approach for converting a mouse antibody into a formsuitable for use in humans is technology practiced commercially byKaloBios Pharmaceuticals, Inc. (Palo Alto, Calif.). This technologyinvolves the use of a proprietary human “acceptor” library to produce an“epitope focused” library for antibody selection.

Another exemplary approach for modifying a mouse antibody into a formsuitable for medical use in humans is HUMAN ENGINEERING™ (HE™)technology, which is practiced commercially by XOMA (US) LLC. See, e.g.,International Application Publication No. WO 93/11794 and U.S. Pat. Nos.5,766,886; 5,770,196; 5,821,123; and 5,869,619.

Any suitable approach, including any of the above approaches, can beused to reduce or eliminate human immunogenicity of a binding protein ofinterest.

In addition, it is possible to create fully human antibodies in mice. Inthis approach, human antibodies are prepared using a transgenic mouse inwhich the mouse's antibody-producing genes have been replaced by asubstantial portion of the human antibody producing genes. Such miceproduce human immunoglobulin instead of murine immunoglobulin molecules.See, e.g., WO 98/24893 (Jacobovitz et al.) and Mendez et al. (1997)NATURE GENETICS15: 146-156. Fully human anti-HGF monoclonal antibodiescan be produced using the following approach. Transgenic mice containinghuman immunoglobulin genes are immunized with the antigen of interest,e.g., HGF. Lymphatic cells from the mice then are obtained from themice, which are then fused with a myeloid-type cell line to prepareimmortal hybridoma cell lines. The hybridoma cell lines are screened andselected to identify hybridoma cell lines that produce antibodiesspecific to HGF.

Binding proteins of the invention can be conjugated with othermolecules, depending upon their intended use. For example, if thebinding protein is going to be used as a therapeutic, then the bindingprotein can be conjugated with another agent, for example, an effectormolecule that modulates or otherwise promotes the therapy. To the extentthat the effector is non-protein based agent, for example, a smallmolecule drug, a radiolabel or toxin, then, the agent can be chemicallycoupled to the binding protein using standard in vitro couplingchemistries. If, on the other hand, the effector molecule is a proteinor peptide, for example, an enzyme, receptor, toxin, growth factor,cytokine or other immunomodulator, then the binding protein can eitherbe chemically coupled to the effector using in vitro couplingchemistries or can be coupled to the effector as a fusion protein.Fusion proteins can be constructed and expressed using the techniquessimilar to those discussed in section II.

IV—Use of Binding Proteins

The binding proteins described herein can be used as a diagnostic agentor a therapeutic agent.

(1) Therapeutic Applications

Because the binding proteins of the invention neutralize the activity ofHGF, they can be used in various therapeutic applications. For example,certain binding proteins of the invention are useful in the preventionor treatment of hyperproliferative diseases or disorders, e.g., variousforms of cancer.

The binding proteins can be used to inhibit or reduce the proliferationof tumor cells. In such an approach, the tumor cells are exposed to atherapeutically effective amount of the binding protein so as to inhibitor reduce proliferation of the tumor cell. In certain embodiments, thebinding proteins inhibit tumor cell proliferation by at least 50%, 60%,70%, 80%, 90%, 95% or 100%.

In certain embodiments, the binding protein is used to inhibit or reduceproliferation of a tumor cell wherein the binding protein reduces theability of hHGF to bind to c-Met. In other embodiments, the bindingprotein is used to inhibit or reduce the proliferation of a tumor celleven when the binding protein binds hHGF but does not substantiallyinhibit hHGF binding to c-Met, as shown by antibody 3B6 in Tables 5 and6.

In addition, the binding protein can be used to inhibit, or slow downtumor growth or development in a mammal. In such a method, an effectiveamount of the binding protein is administered to the mammal so as toinhibit or slow down tumor growth in the mammal. Accordingly, thebinding proteins can be used to treat tumors, for example, in a mammal.The method comprises administering to the mammal a therapeuticallyeffective amount of the binding protein. The binding protein can beadministered alone or in combination with another pharmaceuticallyactive molecule, so as to treat the tumor.

It is contemplated that the binding proteins of the invention can beused in the treatment of a variety of HGF responsive disorders,including, for example, HGF responsive tumor cells in lung cancer,breast cancer, colon cancer, prostate cancer, ovarian cancer, head andneck cancer, ovarian cancer, multiple myeloma, liver cancer, gastriccancer, esophageal cancer, kidney cancer, nasopharangeal cancer,pancreatic cancer, mesothelioma, melanoma and glioblastoma.

As used herein, “treat, “treating” and “treatment” refer to thetreatment of a disease-state in a mammal, particularly in a human, andinclude: (a) preventing the disease-state from occurring in a mammal, inparticular, when such mammal is predisposed to the disease-state but hasnot yet been diagnosed as having it; (b) inhibiting the disease-state,i.e., arresting its development; and/or (c) relieving the disease-state,i.e., causing regression of the disease state.

Generally, a therapeutically effective amount of active component willbe in the range of from about 0.1 mg/kg to about 100 mg/kg, optionallyfrom about 1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to10 mg/kg. The amount administered will depend on variables such as thetype and extent of disease or indication to be treated, the overallhealth status of the particular patient, the relative biologicalefficacy of the binding protein delivered, the formulation of thebinding protein, the presence and types of excipients in theformulation, and the route of administration. The initial dosageadministered may be increased beyond the upper level in order to rapidlyachieve the desired blood-level or tissue level, or the initial dosagemay be smaller than the optimum and the daily dosage may beprogressively increased during the course of treatment depending on theparticular situation. Human dosage can be optimized, e.g., in aconventional Phase I dose escalation study designed to run from 0.5mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors suchas route of administration, dosage amount and the disease conditionbeing treated. Exemplary dosing frequencies are once per day, once perweek and once every two weeks. A preferred route of administration isparenteral, e.g., intravenous infusion. Formulation of monoclonalantibody-based drugs is within ordinary skill in the art. In someembodiments of the invention, the binding protein, e.g., monoclonalantibody, is lyophilized and reconstituted in buffered saline at thetime of administration.

The binding proteins may be administered either alone or in combinationwith other pharmaceutically active ingredients. The other activeingredients, e.g., immunomodulators, can be administered together withthe binding protein, or can be administered before or after the bindingprotein.

Formulations containing the binding proteins for therapeutic use,typically include the binding proteins combined with a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” means buffers, carriers, and excipients, that are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The carrier(s) shouldbe “acceptable” in the sense of being compatible with the otheringredients of the formulations and not deleterious to the recipient.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all buffers, solvents, dispersion media, coatings,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is known in the art.

The formulations can be conveniently presented in a dosage unit form andcan be prepared by any suitable method, including any of the methodswell known in the pharmacy art. A pharmaceutical composition of theinvention should be formulated to be compatible with its intended routeof administration. Examples of routes of administration includeparenteral administration or non-parenteral administration, for example,intravenous, intradermal, inhalation, transdermal (topical),transmucosal, and rectal administration. Useful solutions for oral orparenteral administration can be prepared by any of the methods wellknown in the pharmaceutical art, described, for example, in Remington'sPharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

Formulations suitable for oral administration can be in the form of:discrete units such as injectables, capsules, gelatin capsules, sachets,tablets, troches, or lozenges, each containing a predetermined amount ofthe binding protein; a powder or granular composition; a solution or asuspension in an aqueous liquid or non-aqueous liquid; or anoil-in-water emulsion or a water-in-oil emulsion.

Formulations suitable for parenteral administration include, forexample, the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

In general, compositions suitable for injectable use include aqueoussolutions (where water soluble) or dispersions and powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor ELTM (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). It should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol), and suitablemixtures thereof.

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, sterilization usingthis method can be conducted prior to or following lyophilization andreconstitution. Once the pharmaceutical composition has been formulated,it can be stored, for example, in vials as a solution, suspension, gel,emulsion, solid, or as a dehydrated or lyophilized powder.

(2) Diagnostic Applications

Whenever the binding proteins are used for diagnostic purposes, eitherin vitro or in vivo, the binding proteins typically are labeled eitherdirectly or indirectly with a detectable moiety. The detectable moietycan be any moiety which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³Hydrogen (³H), ¹⁴Carbon (¹⁴C), ³²Phosphorus(³²P), ³⁵Sulfur (³⁵S), or ¹²⁵Iodine (¹²⁵I); a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; an enzyme, such as alkaline phosphatase,beta-galactosidase, or horseradish peroxidase; a spin probe, such as aspin label; or a colored particle, for example, a latex or goldparticle. It is understood that the binding protein can be conjugated tothe detectable moiety using a number of approaches known in the art, forexample, as described in Hunter et al. (1962) NATURE 144: 945; David etal. (1974) BIOCHEMISTRY 13: 1014; Pain et al. (1981) J. IMMUNOL. METH.40: 219; and Nygren (1982) J. HISTOCHEM. AND CYTOCHEM. 30: 407. Thelabels may be detected, e.g., visually or with the aid of aspectrophotometer or other detector.

The binding proteins can be employed in a wide range of immunoassaytechniques available in the art. Exemplary immunoassays include, forexample, sandwich immunoassays, competitive immunoassays,immunohistochemical procedures.

In a sandwich immunoassay, two antibodies that bind an analyte orantigen of interest are used, e.g., one immobilized onto a solidsupport, and one free in solution and labeled with a detectable moiety.When a sample containing the antigen is introduced into this system, theantigen binds to both the immobilized antibody and the labeled antibody,to form a “sandwich” immune complex on the surface of the support. Thecomplexed protein is detected by washing away non-bound samplecomponents and excess labeled antibody, and measuring the amount oflabeled antibody complexed to protein on the support's surface.Alternatively, the antibody free in solution can be detected by a thirdantibody labeled with a detectable moiety which binds the free antibody.A detailed review of immunological assay design, theory and protocolscan be found in numerous texts, including Butt, ed., (1984) PRACTICALIMMUNOLOGY, Marcel Dekker, New York; Harlow et al. eds. (1988)ANTIBODIES, A LABORATORY APPROACH, Cold Spring Harbor Laboratory; andDiamandis et al., eds. (1996) IMMUNOASSAY, Academic Press, Boston.

It is contemplated that the labeled binding proteins are useful as invivo imaging agents, whereby the binding proteins can target the imagingagents to particular tissues of interest in the recipient. A preferredremotely detectable moiety for in vivo imaging includes the radioactiveatom Technetium^(−99m) (^(99m)Tc), a gamma emitter with a half-life ofabout six hours. Non-radioactive moieties also useful in in vivo imaginginclude nitroxide spin labels as well as lanthanide and transition metalions all of which induce proton relaxation in situ. In addition toimmunoimaging, the complexed radioactive moieties may be used instandard radioimmunotherapy protocols to destroy the targeted cell.Preferred nucleotides for high dose radioimmunotherapy include theradioactive atoms ⁹⁰Yttrium (⁹⁰Yt), ¹³¹Iodine (¹³¹I) and ¹¹¹Indium(¹¹¹In). The binding protein can be labeled with ¹³¹I, ¹¹¹In and^(99m)TC using coupling techniques known in the imaging arts. Similarly,procedures for preparing and administering the imaging agent as well ascapturing and processing images are well known in the imaging art and soare not discussed in detail herein. Similarly, methods for performingantibody-based immunotherapies are well known in the art. See, forexample, U.S. Pat. No. 5,534,254.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where processes are described as having,including, or comprising specific process steps, the processes alsoconsist essentially of, or consist of, the recited processing steps.Except where indicated otherwise, the order of steps or order forperforming certain actions are immaterial so long as the inventionremains operable. Moreover, unless otherwise noted, two or more steps oractions may be conducted simultaneously.

EXAMPLES

The following Examples discuss the production and characterization of anumber of anti-hHGF monoclonal antibodies.

Example 1 Production of Anti-hHGF Monoclonal Antibodies

This Example describes the production of a number of anti-hHGFmonoclonal antibodies.

Immunizations, fusions, and primary screens were conducted at MBS Inc.(Portland, Me.), following the Repetitive Immunization Multiple Sites(RIMMS) protocol. Five AJ mice and Five Balb/c mice were immunized withrecombinant human HGF (R&D Systems, Minneapolis, Minn.; Catalog No.294-HGN-025). Two mice with sera displaying highest anti-HGF activity byEnzyme Linked Immunosorbent Assay (ELISA) were chosen for subsequentfusion. Spleens and lymph nodes from the appropriate mice wereharvested. B-cells then were harvested and fused with an myeloma line.Fusion products were serially diluted on one or more plates to nearclonality. Supernatants from the resulting fusions were screened fortheir binding to hHGF by ELISA. Supernatants identified as containingantibodies to HGF were further characterized by in vitro functionaltesting as discussed in the following examples. A panel of hybridomaswas selected and the hybridomas were subcloned and expanded. Themonoclonal antibodies then were purified by affinity chromatography onProtein A/G resin under standard conditions.

Example 2 Sequence Analysis of Anti-hHGF Monoclonal Antibodies

This Example describes isotype and sequence analyses of the anti-hHGFmonoclonal antibodies produced in Example 1.

a. Determination of HGF Murine Monoclonal Antibody Isotypes

The light-chain type and heavy chain isotype of each monoclonal antibodywere determined using the IsoStrip Mouse Monoclonal Antibody IsotypingKit in accordance the manufacturer's instructions (Roche AppliedScience).

All the antibodies were determined to contain a Kappa immunoglobulinlight chain and an IgG1 immunoglobulin heavy chain.

b. Determination of Nucleotide Sequences Encoding Immunoglobulin Heavyand Light Chain Variable Regions

Total RNA was extracted from each monoclonal hybridoma cell line usingthe RNeasy Miniprep kit according to the manufacturer's instructions(Qiagen Venlo, The Netherlands). Full-length first strand cDNA wasgenerated using the BD SMART™ RACE cDNA Amplification Kit according tothe manufacturer's instructions (Clontech) using the oligonucleotideprimers BD SMART II A (5′ aagcagtggtatcaacgcagagtacgcggg 3′) (SEQ ID NO.85) and 5′-RACE CDS Primer (5′ tttttttttttttttttttttttttvn 3′, wherev=a, g, or c and n=a, g, c, or t) (SEQ ID NO. 86) for the purpose of 5′RACE (Rapid Amplification of cDNA Ends).

The variable regions of the Kappa and Heavy (IgG1) immunoglobulin chainswere amplified by PCR (Polymerase Chain Reaction) using the ExpandHigh-Fidelity PCR System (Roche Applied Science) according to themanufacturer's instructions. Heavy chain variable regions were amplifiedwith the 5′ oligonucleotide primer mix Universal Primer Mix A (mix of 5′ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3′ (SEQ ID NO. 87) and 5′ctaatacgactcactatagggc 3′(SEQ ID NO. 88)) and a 3′ IgG1 Constant Regionspecific primer, either 5′ tatgcaaggcttacaaccaca 3′ (SEQ ID NO. 89) or5′ gccagtggatagacagatgggggtgtcg 3′ (SEQ ID NO. 90). Kappa chain variableregions were amplified with the 5′ oligonucleotide primer mix UniversalPrimer Mix A and a 3′ Kappa Constant Region specific primer, either 5′ctcattcctgttgaagctcttgacaat 3′ (SEQ ID NO. 91) or 5′cgactgaggcacctccagatgtt 3′ (SEQ ID NO. 92).

Individual PCR products were fractionated by agarose gel electrophoresisand purified using the Qiaquick Gel Purification kit according to themanufacturer's instructions (Qiagen). The PCR products were subsequentlycloned into the pCR2.1 TOPO plasmid using the topoisomerase basedcloning kit TOPO TA Cloning® Kit (with pCR®2.1-TOPO® vector) accordingto the manufacturer's instructions (Invitrogen, Carlsbad, Calif.) andtransformed into DH5 bacteria using standard transformation techniques.Plasmid DNA isolated from transformed bacterial clones was sequencedusing T7 (5′ TAATACGACTCACTATAGGG 3′) (SEQ ID NO. 93), M13 Forward (5′GTAAAACGACGGCCAGT 3′) (SEQ ID NO. 94), and M13 Reverse primers (5′CAGGAAACAGCTATGACC 3′) (SEQ ID NO. 95) by Agencourt Bioscience usingstandard dideoxy DNA sequencing methods to identify the sequence of thevariable region sequences. The sequences were analyzed using Vector NTIsoftware (Invitrogen, Carlsbad, Calif.) and the IMGT/V-Quest webserver(http://imgt.cines.fr/textes/vquest) to identify and confirm variableregion sequences.

c. Determination of Nucleotide Sequences Encoding Immunoglobulin Heavyand Light Chain Constant Region Sequences for 1A3, 1D3, 1F3, and 2B8Kappa and IgG1 Chains

Full Length cDNAs for the 1A3, 1D3, and 1F3 IgG1 chains were PCRamplified from the cDNA created above using the forward primer

5′ ggggacaagtttgtacaaaaaagcaggctgccaccatgaactttgggctcagattgattttcc 3′(start codon underlined) (SEQ ID NO. 96) and the reverse primer 5′ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagagg 3′ (stop codonunderlined) (SEQ ID NO. 97). Full Length cDNA for the 2B8 IgG1 chain wasamplified from the cDNA created above using the forward primer5′ ggggacaagtttgtacaaaaaagcaggctgccaccatgggatggagctatatcatcctcttt 3′(start codon underlined) (SEQ ID NO. 98) and reverse primer5′ ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagag 3′ (stop codonunderlined) (SEQ ID NO. 99).

Full Length cDNA for the 2B8 Kappa Chain was amplified using the forwardprimer 5′ ggggacaagtttgtacaaaaaagcaggctgccaccatggaatcacagactctggtcttcata3′ (start codon underlined) (SEQ ID NO. 100) and the reverse primer

5′ ggggaccactttgtacaagaaagctgggt ctaacactcattcctgttgaagctc 3′ (stopcodon underlined) (SEQ ID NO. 101). PCR fragments were subcloned intopDONR221 (Invitrogen, Carlsbad, Calif.) by Gateway BP recombinationreaction (Invitrogen, Carlsbad, Calif.) and sequenced by AgencourtBioscience using standard dideoxy DNA sequencing methods to identify thesequence of the constant region and further confirm variable regionsequences.

d. Sequence Analysis

Variable Regions (normal text) were identified using IMGT/V-QUESTwebserver software (http://imgt.cines.fr/textes/vquest/). Signal Peptidesequences were predicted based on identification of the in frame startcodon (ATG) that was upstream of the identified Variable Region. SignalPeptide sequences were identified and are underlined below.

The last nucleotide of each variable region is the first base of thenext codon generated by the variable/constant region junction. Thisnucleotide is included in the variable region because it is part of thatexon. Amino acid sequences of the constant regions listed below includethe translation of this junction codon.

In order to create the complete heavy or kappa chain antibody sequences,the variable region sequences noted below are combined with theirrespective constant region sequences (the signal sequences areunderlined).

(1) 1A3 Heavy Chain Variable Region (SEQ ID NO. 1)

  1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc121 tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca181 gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca241 gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc421 tcag

(2) 1A3 Kappa Light Chain Variable Region (SEQ ID NO. 3)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc121 atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag181 ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct301 gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg361 gggaccaagc tggaaataaa ac

(3) 2B8 Heavy Chain Variable Region (SEQ ID NO. 11)

  1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag 61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct181 ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat241 gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg301 caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt361 ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc ag

(4) 2B8 Kappa Light Chain Variable Region (SEQ ID NO. 13)

  1 atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg 61 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat241 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct301 gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg361 gggaccaggc tggaaataaa ac

(5) 2F8 Heavy Chain Variable Region (SEQ ID NO. 21)

  1 atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag 61 gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc121 tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct181 ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat241 gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatg301 cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg361 ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc ag

(6) 2F8 Kappa Light Chain Variable Region (SEQ ID NO. 23)

  1 atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt 61 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc121 atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac181 caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct241 gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat301 cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc361 acgttcggtg ctgggaccaa gctggagctg aaac

(7) 3B6 Heavy Chain Variable Region (SEQ ID NO. 31)

  1 atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 61 gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc121 tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct181 ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat241 ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg301 cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg361 ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcag

(8) 3B6 Kappa Light Chain Variable Region (2 Possible ATG Start Codons(Uppercase)) (SEQ ID NO. 33)

  1 ATGgacATGa ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc 61 aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga121 gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag181 aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc241 ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg301 gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc361 ggagggggga ccaagctgga aataaagc

(9) 3D11 Heavy Chain Variable Region (SEQ ID NO. 41)

  1 atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag 61 gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact121 tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca181 ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg241 tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa301 atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct361 tactggggcc aagggactct ggtcactgtc tctgcag

(10) 3D11 Kappa Light Chain Variable Region (SEQ ID NO. 43)

  1 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc 61 agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag121 gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag181 tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct241 gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag301 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt361 gctgggacca agctggagct gaaac

(11) 1D3 Heavy Chain Variable Region (SEQ ID NO. 51)

  1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc121 tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca241 gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg301 caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat361 ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc421 tcag

(12) 1D3 Kappa Light Chain Variable Region (SEQ ID NO. 53)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt 61 gacatccaga tgactcagtc tccagectec ctatctgtat ctgtgggaga aactgtcacc121 atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag181 ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct301 gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg361 gggaccaaac tggaaataaa ac

(13) 1F3 Heavy Chain Variable Region (SEQ ID NO. 61)

  1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag 61 gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc121 tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca181 gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca241 gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc421 tcag

(14) 1F3 Kappa Light Chain Variable Region (SEQ ID NO. 63)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc121 atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag181 ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg361 gggaccagac tggaaattaa ac

(15) 3A12 Heavy Chain Variable Region (SEQ ID NO. 71)

  1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa 61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc121 tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca241 gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg301 caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat361 ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc421 tcag

(16) 3A12 Kappa Light Chain Variable Region (SEQ ID NO. 73)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc121 atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag181 ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg361 gggaccaaac tagaaataaa ac

(17) Reference Mouse IgG1 Heavy Chain Constant Region (J00453) (SEQ IDNO. 81)

  1 ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 61 ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct121 ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg gagtctgacc181 tctacactct gagcagctca gtgactgtcc cctccagccc tcggcccagc gagaccgtca241 cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg301 attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc361 ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg421 tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg481 tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca541 gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca601 acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga661 aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca721 gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga781 atgggcagcc agcggagaac tacaagaaca ctcagcccat catgaacacg aatggctctt841 acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca901 cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact961 ctcctggtaa atga

(18) Mouse IgG1 Heavy Chain Constant Region Determined for 1A3, 1D3,1F3, and 2B8 (Derived from AJ Strain Mice) (SEQ ID NO. 82)

  1 ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 61 ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct121 ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg cagtctgacc181 tctacactct gagcagctca gtgactgtcc cctccagcac ctggcccagc gagaccgtca241 cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg301 attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc361 ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg421 tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg481 tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca541 gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca601 acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga661 aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca721 gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga781 atgggcagcc agcggagaac tacaagaaca ctcagcccat catggacaca gatggctctt841 acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca901 cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact961 ctcctggtaa atga

(19) Reference Mouse Kappa Light Chain Constant Region (V00807) andMouse Kappa Light Chain Constant Region Determined for 1D3, 1F3, and 2B8(Derived from AJ Strain Mice) (SEQ ID NO. 83)

  1 gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg 61 gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt121 ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca181 gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac gagtatgaac241 gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga301 gcttcaacag gaatgagtgt tag

(20) Mouse Kappa Light Chain Constant Region Determined for 1A3Containing One Altered Nucleotide Compared to 1D3, 1F3, and 2B8(Underlined) (SEQ ID NO. 84)

  1 gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg 61 gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt121 ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca181 gcaaagacag cacctacagc atgagcagca ccctcatgtt gaccaaggac gagtatgaac241 gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga301 gcttcaacag gaatgagtgt tag

Each of the amino acid sequences defining the immunoglobulin heavy chainvariable regions for the antibodies produced in Example 1 are set forthin FIG. 2. Each of the sequences are aligned with one another and thesequences defining the signal peptide, CDR₁, CDR₂ and CDR₃ areidentified by boxes. FIG. 3 shows an alignment of the separate CDR₁,CDR₂ and CDR₃ sequences for each of the antibodies.

Each of the amino acid sequences defining the immunoglobulin light chainvariable regions for each of the antibodies produced in Example 1 areset forth in FIG. 4. Each of the sequences are aligned with one anotherand the sequences defining the signal peptide, CDR₁, CDR₂ and CDR₃ areidentified by boxes. FIG. 5 shows an alignment of the separate CDR₁,CDR₂ and CDR₃ sequences for each of the antibodies.

For convenience, Table 1 provides a concordance chart showing thecorrespondence between the antibody sequences discussed in this Examplewith those presented in the Sequence Listing.

TABLE 1 SEQ. ID NO. Protein or Nucleic Acid 1 Heavy Chain VariableRegion 1A3 - nucleic acid 2 Heavy Chain Variable Region 1A3 - protein 3Light (kappa) Chain Variable Region 1A3 - nucleic acid 4 Light (kappa)Chain Variable Region 1A3 - protein 5 Heavy Chain CDR₁ 1A3 6 Heavy ChainCDR₂ 1A3 7 Heavy Chain CDR₃ 1A3 8 Light (kappa) Chain CDR₁ 1A3 9 Light(kappa) Chain CDR₂ 1A3 10 Light (kappa) Chain CDR₃ 1A3 11 Heavy ChainVariable Region 2B8 - nucleic acid 12 Heavy Chain Variable Region 2B8 -protein 13 Light (kappa) Chain Variable Region 2B8 - nucleic acid 14Light (kappa) Chain Variable Region 2B8 - protein 15 Heavy Chain CDR₁2B8 16 Heavy Chain CDR₂ 2B8 17 Heavy Chain CDR₃ 2B8 18 Light (kappa)Chain CDR₁ 2B8 19 Light (kappa) Chain CDR₂ 2B8 20 Light (kappa) ChainCDR₃ 2B8 21 Heavy Chain Variable Region 2F8 - nucleic acid 22 HeavyChain Variable Region 2F8 - protein 23 Light (kappa) Chain VariableRegion 2F8 - nucleic acid 24 Light (kappa) Chain Variable Region 2F8 -protein 25 Heavy Chain CDR₁ 2F8 26 Heavy Chain CDR₂ 2F8 27 Heavy ChainCDR₃ 2F8 28 Light (kappa) Chain CDR₁ 2F8 29 Light (kappa) Chain CDR₂ 2F830 Light (kappa) Chain CDR₃ 2F8 31 Heavy Chain Variable Region 3B6 -nucleic acid 32 Heavy Chain Variable Region 3B6 - protein 33 Light(kappa) Chain Variable Region 3B6 - nucleic acid 34 Light (kappa) ChainVariable Region 3B6 - protein 35 Heavy Chain CDR₁ 3B6 36 Heavy ChainCDR₂ 3B6 37 Heavy Chain CDR₃ 3B6 38 Light (kappa) Chain CDR₁ 3B6 39Light (kappa) Chain CDR₂ 3B6 40 Light (kappa) Chain CDR₃ 3B6 41 HeavyChain Variable Region 3D11 - nucleic acid 42 Heavy Chain Variable Region3D11 - protein 43 Light (kappa) Chain Variable Region 3D11- nucleic acid44 Light (kappa) Chain Variable Region 3D11 - protein 45 Heavy ChainCDR₁ 3D11 46 Heavy Chain CDR₂ 3D11 47 Heavy Chain CDR₃ 3D11 48 Light(kappa) Chain CDR₁ 3D11 49 Light (kappa) Chain CDR₂ 3D11 50 Light(kappa) Chain CDR₃ 3D11 51 Heavy Chain Variable Region 1D3 - nucleicacid 52 Heavy Chain Variable Region 1D3 - protein 53 Light (kappa) ChainVariable Region 1D3 - nucleic acid 54 Light (kappa) Chain VariableRegion 1D3 - protein 55 Heavy Chain CDR₁ 1D3 56 Heavy Chain CDR₂ 1D3 57Heavy Chain CDR₃ 1D3 58 Light (kappa) Chain CDR₁ 1D3 59 Light (kappa)Chain CDR₂ 1D3 60 Light (kappa) Chain CDR₃ 1D3 61 Heavy Chain VariableRegion 1F3 - nucleic acid 62 Heavy Chain Variable Region 1F3 - protein63 Light (kappa) Chain Variable Region 1F3 - nucleic acid 64 Light(kappa) Chain Variable Region 1F3 - protein 65 Heavy Chain CDR₁ 1F3 66Heavy Chain CDR₂ 1F3 67 Heavy Chain CDR₃ 1F3 68 Light (kappa) Chain CDR₁1F3 69 Light (kappa) Chain CDR₂ 1F3 70 Light (kappa) Chain CDR₃ 1F3 71Heavy Chain Variable Region 3A12 - nucleic acid 72 Heavy Chain VariableRegion 3A12 - protein 73 Light (kappa) Chain Variable Region 3A12 -nucleic acid 74 Light (kappa) Chain Variable Region 3A12 - protein 75Heavy Chain CDR₁ 3A12 76 Heavy Chain CDR₂ 3A12 77 Heavy Chain CDR₃ 3A1278 Light (kappa) Chain CDR₁ 3A12 79 Light (kappa) Chain CDR₂ 3A12 80Light (kappa) Chain CDR₃ 3A12

Also, for convenience, the following sequences represent the actual orcontemplated full length heavy and light chain sequences (i.e.,containing both the variable and constant region sequences) for each ofthe antibodies described in this Example. It is noted that the constantregions of the murine antibodies 2F8, 3A12, 3B6, and 3D11 were notsequenced but are presumed to have the same constant region sequences asthe 1D3, 1F3, and 2B8 antibodies, which were sequenced, as they were allderived from AJ strain mice. It is appreciated, however, that thevariable region sequences described herein can be ligated to each of anumber of other constant region sequences known to those skilled in theart to produce active full length immunoglobulin heavy and light chains.

(1) Nucleic Acid Sequence Encoding the Full Length 1A3 Heavy ChainSequence (1A3 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 122)

   1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa  61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 121 tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca 181 gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca 241 gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat 361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc1381 cactctcctg gtaaatga

(2) Protein Sequence Defining the Full Length 1A3 Heavy Chain Sequence(1A3 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 123)

  1 evqlvesggg lvqpggslkl scaaseftfs nyymswvrqt pekrlqwvay ispgggssyy 61 pasvkgrfti srdnakntly lqmsslksed tamyycarqg dgyygdyamd ywgqgtsvtv121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn421 tftcsvlheg lhnhhteksl shspgk

(3) Nucleic Acid Sequence Encoding the Full Length 1A3 Light ChainSequence (1A3 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 124)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc121 atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag181 ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct301 gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg361 gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcatg601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(4) Protein Sequence Defining the Full Length 1A3 Light Chain Sequence(1A3 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 125)

  1 diqmtqspas lsysvgetvt itcraseniy snlawyqqkq gkspqllvya atnladgvps 61 rfsgsgsgtq fslkinslqs edfgtyycqh fwgtpytfgg gtkleikrad aaptvsifpp121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlm181 ltkdeyerhn sytceathkt stspivksfn rnec

(5) Nucleic Acid Sequence Encoding the Full Length 2B8 Heavy ChainSequence (2B8 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 126)

   1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag  61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc 121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct 181 ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat 241 gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 301 caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt 361 ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg 421 acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 481 accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 541 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 601 ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 661 gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 721 tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 781 cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 841 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 901 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 961 cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca1021 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca1081 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc1141 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag1201 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct1321 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt1381 aaatga

(6) Protein Sequence Defining the Full Length 2B8 Heavy Chain Sequence(2B8 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 127)

  1 qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny 61 nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gttltvssak121 ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsdly181 tlsssvtvps stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvfifpp241 kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsvse301 lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl361 tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks nweagntftc421 svlheglhnh htekslshsp gk

(7) Nucleic Acid Sequence Encoding the Full Length 2B8 Light ChainSequence (2B8 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 128)

  1 atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg 61 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat241 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct301 gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg361 gggaccaggc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(8) Protein Sequence Defining the Full Length 2B8 Light Chain Sequence(2B8 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 129)

  1 nivmtqspks msmsvgervt lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd 61 rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrad aaptvsifpp121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt181 ltkdeyerhn sytceathkt stspivksfn rnec

(9) Nucleic Acid Sequence Encoding the Full Length 2F8 Heavy ChainSequence (2F8 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 130)

   1 atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag  61 gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc 121 tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct 181 ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat 241 gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatg 301 cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg 361 ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg 421 acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 481 accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 541 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 601 ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 661 gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 721 tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 781 cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 841 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 901 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 961 cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca1021 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca1081 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtdtgacc1141 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag1201 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc1261 tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct1321 gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt1381 aaatga

(10) Protein Sequence Defining the Full Length 2F8 Heavy Chain Sequence(2F8 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 131)

  1 qvqlkqsgae lvrpgtsvkm sckasgytft tyyihwvnqr pgqglewigk igpgsgstyy 61 nemfkdkatl tvdtssstay mqlssltsdd savyfcarrg lgrgfdywgq gttltvssak121 ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsdly181 tlsssvtvps stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvfifpp241 kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsvse301 lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl361 tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks nweagntftc421 svlheglhnh htekslshsp gk

(11) Nucleic Acid Sequence Encoding the Full Length 2F8 Light ChainSequence (2F8 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 132)

  1 atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt 61 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc121 atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac181 caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct241 gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat301 cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc361 acgttcggtg ctgggaccaa gctggagctg aaacgggctg atgctgcacc aactgtatcc421 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg481 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa541 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc601 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc661 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag

(12) Protein Sequence Defining the Full Length 2F8 Light Chain Sequence(2F8 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 133)

  1 divltqspas lavslgqrat isckasqsvd ydgnsyinwy qqkpgqppkv liyvasnles 61 giparfsgsg sgtdftlnih pveeedaaty ycqqsiedpp tfgagtklel kradaaptvs121 ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq ngvlnswtdq dskdstysms181 stltltkdey erhnsytcea thktstspiv ksfnrnec

(13) Nucleic Acid Sequence Encoding the Full Length 3B6 Heavy ChainSequence (3B6 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 134)

   1 atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag  61 gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc 121 tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct 181 ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat 241 ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg 301 cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg 361 ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc 421 aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 481 atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg 541 aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc 601 tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc 661 tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat 721 tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc 781 ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 841 gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg 901 cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt 961 gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac1021 agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag1081 gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt1141 ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat1201 gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac1261 ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc1321 tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct1381 cctggtaaat ga

(14) Protein Sequence Defining the Full Length 3B6 Heavy Chain Sequence(3B6 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 135)

  1 qvqlqqsgae lvrpgssvki sckasgyvfs sywmnwvkqr pgqglewigq iypgdgdsny 61 ngnfkgkatl tadkssstay mqlssltsed savyfcasql glrenyfdyw gqgttltvss121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv301 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf421 tcsvlheglh nhhtekslsh spgk

(15) Nucleic Acid Sequence Encoding the Full Length 3B6 Light ChainSequence (3B6 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 136)

  1 ATGgacATGa ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc 61 aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga121 gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag181 aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc241 ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg301 gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc361 ggagggggga ccaagctgga aataaagcgg gctgatgctg caccaactgt atccatcttc421 ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac481 ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc541 gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc601 ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac661 aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgtta g

(16) Protein Sequence Defining the Full Length 3B6 Light Chain Sequence(3B6 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 137)

  1 dikmtqspss myaslgervt itckasqdik sylswfqqkp gkspktliyr vnrlvdgvps 61 rfsgsgsgqd ssltitslen edmgiyyclq ydefpftfgg gtkleikrad aaptvsifpp121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt181 ltkdeyerhn sytceathkt stspivksfn rnec

(17) Nucleic Acid Sequence Encoding the Full Length 3D11 Heavy ChainSequence (3D11 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 138)

   1 atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag  61 gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact 121 tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca 181 ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg 241 tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa 301 atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct 361 tactggggcc aagggactct ggtcactgtc tctgcagcca aaacgacacc cccatctgtc 421 tatccactgg cccctggatc tgctgcccaa actaactcca tggtgaccct gggatgcctg 481 gtcaagggct atttccctga gccagtgaca gtgacctgga actctggatc cctgtccagc 541 ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acactctgag cagctcagtg 601 actgtcccct ccagcacctg gcccagcgag accgtcacct gcaacgttgc ccacccggcc 661 agcagcacca aggtggacaa gaaaattgtg cccagggatt gtggttgtaa gccttgcata 721 tgtacagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa ggatgtgctc 781 accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc 841 gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc 901 cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag 961 gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt ccctgccccc1021 atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg ctccacaggt gtacaccatt1081 ccacctccca aggagcagat ggccaaggat aaagtcagtc tgacctgcat gataacagac1141 ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc ggagaactac1201 aagaacactc agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat1261 gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt acatgagggc1321 ctgcacaacc accatactga gaagagcctc tcccactctc ctggtaaatg a

(18) Protein Sequence Defining the Full Length 3D11 Heavy Chain Sequence(3D11 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 139)

  1 qvqlkesgpg lvapsqslsi tctvsgfslt syslhwvrqp pgkglewlgv iwaggntnyn 61 sslmsrltir kdnsksqvfl kmnslqtddt amyycarerf aywgqgtlvt vsaakttpps121 vyplapgsaa qtnsmvtlgc lvkgyfpepv tvtwnsgsls sgvhtfpavl qsdlytlsss181 vtvpsstwps etvtcnvahp asstkvdkki vprdcgckpc ictvpevssv fifppkpkdv241 ltitltpkvt cvvvdiskdd pevqfswfvd dvevhtaqtq preeqfnstf rsvselpimh301 qdwlngkefk crvnsaafpa piektisktk grpkapqvyt ipppkeqmak dkvsltcmit361 dffpeditve wqwngqpaen ykntqpimdt dgsyfvyskl nvqksnweag ntftcsvlhe421 glhnhhteks lshspgk

(19) Nucleic Acid Sequence Encoding the Full Length 3D11 Light ChainSequence (3D11 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 140)

  1 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc 61 agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag121 gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag181 tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct241 gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag301 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt361 gctgggacca agctggagct gaaacgggct gatgctgcac caactgtatc catcttccca421 ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc481 taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc541 ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc601 acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag661 acatcaactt cacccattgt caagagcttc aacaggaatg agtgttag

(20) Protein Sequence Defining the Full Length 3D11 Light Chain Sequence(3D11 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 141)

  1 qivltqspai msaypgekvt mtcsasssys ymhwyqqksg tspkrwiydt sklasgvpar 61 fsgsgsgtsy sltissmeae daatyycqqw ssnpltfgag tklelkrada aptvsifpps121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl181 tkdeyerhns ytceathkts tspivksfnr nec

(21) Nucleic Acid Sequence Encoding the Full Length 1D3 Heavy ChainSequence (1D3 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 142)

   1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa  61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 121 tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca 181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca 241 gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg 301 caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat 361 ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc 421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc1381 cactctcctg gtaaatga

(22) Protein Sequence Defining the Full Length 1D3 Heavy chain sequence(1D3 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 143)

  1 evqlvesggg lvqpggslkl scaasgftfs dyymswvrqt pekrlewvay issgggstyy 61 pdsvkgrfti srdnakntly lqmsslksed taiyycvrqg dgyygdyamd ywgqgtsviv121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn421 tftcsvlheg lhnhhteksl shspgk

(23) Nucleic Acid Sequence Encoding the Full Length 1D3 Light ChainSequence (1D3 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 144)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt 61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc121 atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag181 ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct301 gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg361 gggaccaaac tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(24) Protein Sequence Defining the Full Length 1D3 Light Chain Sequence(1D3 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 145)

  1 diqmtqspas lsvsvgetvt itcrtseniy snlawyqqkq gkspqlliya atnladgvps 61 rfsgsgsgtq fslrinslqs edfgryycqh fwgtpytfgg gtkleikrad aaptvsifpp121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt181 ltkdeyerhn sytceathkt stspivksfn rnec

(25) Nucleic Acid Sequence Encoding the Full Length 1F3 Heavy ChainSequence (1F3 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 146)

   1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag  61 gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc 121 tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca 181 gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca 241 gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg 301 caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat 361 ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc1381 cactctcctg gtaaatga

(26) Protein Sequence Defining the Full Length 1F3 Heavy Chain Sequence(1F3 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 147)

  1 evqlvesggg lvqsggslkl scaasgftfs nyfmswvrqt pekrlewvay issgggstyy 61 pdsvkgrfti srdnakntly lqmsslksed tamyycvrqg dgyygdyamd ywgqgtsvtv121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr301 syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn421 tftcsvlheg lhnhhteksl shspgk

(27) Nucleic Acid Sequence Encoding the Full Length 1F3 Light ChainSequence (1F3 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 148)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc121 atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag181 ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg361 gggaccagac tggaaattaa acgggctgat gctgcaccaa ctgtatccat cttcccacca421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(28) Protein Sequence Defining the Full Length 1F3 Light Chain Sequence(1F3 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 149)

  1 diqmtqspas lsysvgetvt itcraseniy snlawyqqkq gkspqllvyd athlpdgvps 61 rfsgsgsgtq fslkinslqs edfgsyycqh fwgtpytfgg gtrleikrad aaptvsifpp121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt181 ltkdeyerhn sytceathkt stspivksfn rnec

(29) Nucleic Acid Sequence Encoding the Full Length 3A12 Heavy ChainSequence (3A12 Heavy Chain Variable Region and IgG1 Constant Region)(Signal Sequence Underlined) (SEQ ID NO. 150)

   1 atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa  61 gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc 121 tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca 181 gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca 241 gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 301 caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat 361 ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 421 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 481 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 541 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 601 gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 661 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 721 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 781 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 841 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 901 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 961 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg1021 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga1081 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa1141 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag1201 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc1261 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact1321 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc1381 cactctcctg gtaaatga

(30) Protein Sequence Defining the Full Length 3A12 Heavy Chain Sequence(3A12 Heavy Chain Variable Region and IgG1 Constant Region) (withoutSignal Sequence) (SEQ ID NO. 151)

  1 evqlvesggg lvqpggslki scaasgftfs nyfmswvrqt pekrlewvay issgggstyy 61 pdsvkgrfti srdnakntly lqmnslksed tamyycvrqg dgyygdyamd ywgqgtsvtv121 ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq181 sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf241 ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr301 svselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd361 kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn421 tftcsvlheg lhnhhteksl shspgk

(31) Nucleic Acid Sequence Encoding the Full Length 3A12 Light ChainSequence (3A12 Kappa Variable Region and Constant Region) (SignalSequence Underlined) (SEQ ID NO. 152)

  1 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt 61 gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc121 atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag181 ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca241 aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct301 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg361 gggaccaaac tagaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca421 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac481 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg541 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg601 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca661 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag

(32) Protein Sequence Defining the Full Length 3A12 Light Chain Sequence(3A12 Kappa Variable Region and Constant Region) (without SignalSequence) (SEQ ID NO. 153)

  1 diqmtqspas lsvsvgetvt itcraseniy inlawyqqkq gkspqllvha atkladgvps 61 rfsgsgsgtq yslkinslqs edfgsyycqh fwgtpytfgg gtkleikrad aaptvsifpp121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt181 ltkdeyerhn sytceathkt stspivksfn rnec

For convenience, Table 2 provides a concordance chart showing thecorrespondence between the full length sequences of the antibodiesdiscussed in this Example with those presented in the Sequence Listing.

TABLE 2 SEQ. ID NO. Protein or Nucleic Acid 122 1A3 Heavy Variable +IgG1 constant - nucleic acid 123 1A3 Heavy Variable + IgG1 constant -protein 124 1A3 Light Variable + constant - nucleic acid 125 1A3 LightVariable + constant - protein 126 2B8 Heavy Variable + IgG1 constant -nucleic acid 127 2B8 Heavy Variable + IgG1 constant - protein 128 2B8Light Variable + constant - nucleic acid 129 2B8 Light Variable +constant - protein 130 2F8 Heavy Variable + IgG1 constant - nucleic acid131 2F8 Heavy Variable + IgG1 constant - protein 132 2F8 LightVariable + constant - nucleic acid 133 2F8 Light Variable + constant -protein 134 3B6 Heavy Variable + IgG1 constant - nucleic acid 135 3B6Heavy Variable + IgG1 constant - protein 136 3B6 Light Variable +constant - nucleic acid 137 3B6 Light Variable + constant - protein 1383D11 Heavy Variable + IgG1 constant - nucleic acid 139 3D11 HeavyVariable + IgG1 constant - protein 140 3D11 Light Variable + constant -nucleic acid 141 3D11 Light Variable + constant - protein 142 1D3 HeavyVariable + IgG1 constant - nucleic acid 143 1D3 Heavy Variable + IgG1constant - protein 144 1D3 Light Variable + constant - nucleic acid 1451D3 Light Variable + constant - protein 146 1F3 Heavy Variable + IgG1constant - nucleic acid 147 1F3 Heavy Variable + IgG1 constant - protein148 1F3 Light Variable + constant - nucleic acid 149 1F3 LightVariable + constant - protein 150 3A12 Heavy Variable + IgG1 constant -nucleic acid 151 3A12 Heavy Variable + IgG1 constant - protein 152 3A12Light Variable + constant - nucleic acid 153 3A12 Light Variable +constant - protein

Example 3 Production of Various Recombinant hHGF Proteins

This Example describes the cloning and expression of a number ofrecombinant proteins used to characterize the antibodies created inExample 1 and in Example 14. In particular, this Example describes thecloning and expression of recombinant hHGF protein, a recombinant hHGFprotein containing a glycine to glutamate substitution at position 555(G555E), a recombinant hHGF protein containing a cysteine to argininesubstitution at position 561 (C561R), a recombinant mouse-human-mouse(mhm) chimeric HGF protein containing the human V495-L585 HGF sequencedisposed within mouse HGF sequence, a recombinant mhm chimeric HGFprotein containing the human I499-R566 HGF sequence disposed withinmouse HGF sequence, and a recombinant mhm chimeric HGF proteincontaining human W507-L585 HGF sequence disposed within mouse HGFsequence.

The following expression constructs were generated using standardmolecular techniques and the resulting cDNA sequences were confirmed byDNA sequencing:

a. hHGF-Fc

In a first round of PCR, two overlapping PCR fragments were generatedintroducing a Not I site and encoding a 6×His tag between hHGF andhIgFc. The overlapping PCR fragments served as template in a secondround to amplify hHGF-his-IgFc. The resulting fragment was digested byNheI and BamHI and cloned into pcDNA5/FRT (Invitrogen, #35-3014). Then,hHGF was amplified from Invitrogen clone ID: IOH29794 (human HGF cDNA).The sequence was found to correspond to the sequence deposited at theNCBI under accession number NM_(—)000601.4.

(1) 5′ hHGF NheI Primer

ACTGGCTAGCATGTGGGTGACCAAACTCCT (SEQ ID NO. 102)

(2) 3′ hHGF NotI His Tag Primer

GTGATGGTGATGGTGATGGCGGCCGCATGACTGTGGTACCTTATATG (SEQ ID NO. 103)

(3) 5′ HisIgFc Primer

ACTGGCGGCCGCCATCACCATCACCATCAC (SEQ ID NO. 104)

(4) 3′ IgFc BamHI Primer

ACTGGGATCCTCACTATTTACCCGGGGACAG (SEQ ID NO. 105)

b. hHGF-Fc G555E and hHGF-Fc C561R

hHGF-Fc mutants G555E and C561R were generated by site directedmutagenesis using the QuikChange II XL site-directed mutagenesis kit(Stratagene) according to manufacturer's instructions.

(1) hHGF-Fc (G555E) Sense Primer

CATGATGTCCACGAAAGAGGAGATGAG (SEQ ID NO. 106)

(2) hHGF-Fc (G555E) Anti-Sense Primer

CTCATCTCCTCTTTCGTGGACATCATG (SEQ ID NO. 107)

(31 hHGF-Fc (C561R) Sense Primer

GGAAGAGGAGATGAGAAACGCAAACAGGTTCTCAATG (SEQ ID NO. 108)

(4) hHGF-Fc (C561R) Anti-Sense Primer

CATTGAGAACCTGTTTGCGTTTCTCATCTCCTCTTCC (SEQ ID NO. 109)

c. Mouse-Human-Mouse Chimera Fc

The mouse-human-mouse chimera IgFc construct contains mHGF alphachain-hHGF, β-chain amino acids Val 495-Leu 585 of human HGF, and mHGFC-terminal beta chain followed by 6×His tag and IgG-Fc.

Human HGF cDNA encoding amino acids V495-L585 was amplified fromInvitrogen clone ID: IOH29794 (human HGF cDNA). The sequence correspondsto the sequence deposited at the NCBI under accession numberNM_(—)000601.4. Mouse HGF sequences were amplified by RT-PCR from mouseliver total RNA (Clontech, #636603) using the Super Script One StepRT-PCR kit from Invitrogen (#10928-034) according to manufacturer'sinstructions. The mHGF cDNA sequence corresponds to the sequencedeposited at the NCBI under accession number D10213.1.

Three fragments, referred to as Fragments 1, 2, and 3, were generatedusing overlapping PCR primers and annealed in consecutive rounds of PCRamplification. The final product was cleaved with NheI and NotI andcloned into pcDNA5/FRT IgGFc.

(1) Fragment 1 Primers for mHGF Alpha Chain 5′NheI

5′ATCGGCTAGCATGATGTGGGGGACCAAAC (SEQ ID NO. 110)

3′ GAATCCCATTTACAACCCGCAGTTGTTTTGTTTTGG (SEQ ID NO. 111)

(2) Fragment 2 Primers for hHGF Beta Chain aa V495-L585

5′ CCAAAACAAAACAACTGCGGGTTGTAAATGGGATTC (SEQ ID NO. 112)

3′ CAGGATTGCAGGTCGAGCAAGCTTCATTAAAACCAGATCT (SEQ ID NO. 113)

(3) Fragment 3 Primer for mHGF Beta Chain C-Terminus 3′ NotI

5′ AGATCTGGTTTTAATGAAGCTTGCTCGACCTGCAATCCTG (SEQ ID NO. 114)

3′ GTAATTTTGACATACAAGTTGTGCGGCCGCCATCACCATCACCATCAC (SEQ ID NO. 115)

d. Construction of hHGF and mhm Chimera

The vectors encoding hHGF and mhm chimera (V495-L585), pcDNA5/FRT hHGFand pcDNA5/FRT-mhm chimera (V495-L585), without Fc-tag were generated bysite directed mutagenesis. A stop codon was introduced 3′ of the 6×Histag using the QuikChange II XL site-directed mutagenesis kit(Stratagene) according to manufacturer's instructions. The mutagenesisprimer included Primer 1: CATCACCATCACCATCACTAAGCGGGTCTGGTGCCACG (SEQ IDNO. 116), and Primer 2: CGTGGCACCAGACCCGCTTAGTGATGGTGATGGTGATG (SEQ IDNO. 117).

In addition, two additional mhm chimeras were created from thepcDNA51FRT-mhm (V495-L585) construct by site directed mutagenesis usingthe QuikChange II XL site-directed mutagenesis kit (Stratagene)according to manufacturer's instructions. One mhm construct containedthe region of I499-R556 of hHGF disposed between murine sequences. Theother mhm construct contained the region of W507-L585 of hHGF disposedbetween murine sequences.

For the mhm chimera (I499-R556), the following point mutations were madein order in the template pcDNA5/FRT-mhm chimera (V495-L585) construct:D558E, C561R, V564I, V567I and M583L, using the appropriateoligonucleotide sequences. For the mhm chimera (W507-L585), thefollowing point mutations were introduced in one step in the templatepcDNA5/FRT-mhm chimera (V495-L585) construct: Q502R, N504T and I505V,using the appropriate oligonucleotide sequences.

The resulting nucleotide sequence of the hHGF-Fc protein is set forth asSEQ ID NO. 118, including signal sequence (nucleotides 1-93) andprodomain (nucleotides 94-162). The amino acid sequence of the hHGF-Fcprotein is set forth as SEQ ID NO. 119.

The resulting nucleotide sequence encoding the mhm (V495-L585)-Fcchimeric protein is set forth in SEQ ID NO. 120, including signalsequence (nucleotides 1-96) and prodomain (nucleotides 97-165). Theamino acid sequence of the mhm (V495-L585)-Fc chimeric protein is setforth in SEQ ID NO. 121.

The resulting nucleotide sequence encoding, and the protein sequencedefining, the mhm (V495-L585) construct are set forth in SEQ ID NOS. 211and 212, respectively. The nucleic acid sequence set forth in SEQ ID NO.211 includes the signal sequence (nucleotides 1-96) and the prodomain(nucleotides 97-165), and the protein sequence set forth in SEQ ID NO.212 includes the active protein sequence (without the signal sequence orthe prodomain). The resulting nucleotide sequence encoding, and theprotein sequence defining, the mhm (I499-R556) construct are set forthin SEQ ID NOS. 213 and 214, respectively. The nucleic acid sequence setforth in SEQ ID NO. 213 includes the signal sequence (nucleotides 1-96)and the prodomain (nucleotides 97-165), and the protein sequence setforth in SEQ ID NO. 214 includes the active protein sequence (withoutthe signal sequence or the prodomain). The resulting nucleotide sequenceencoding, and the protein sequence defining, the mhm (W507-L585) are setforth in SEQ ID NOS. 215 and 216, respectively. The nucleic acidsequence set forth in SEQ ID NO. 215 includes the signal sequence(nucleotides 1-96) and the prodomain (nucleotides 97-165), and theprotein sequence set forth in SEQ ID NO. 216 includes the active proteinsequence (without the signal sequence or the prodomain)

e. Protein Expression

(1) Cell culture

CHO FlpIn cells (Invitrogen, Catalog No. R758-07)) were grown in F12Kmedia (ATCC, Catalog No. 30-2004), 10% FCS (Invitrogen, Catalog No.10438026), 1% Penicillin (10000 units/mL)/Streptomycin (10,000 μg/mL)(Invitrogen, Catalog No. 15140-122) at 37° C., 5% CO₂, 100 μg/mL Zeocin(Invitrogen, Catalog No. R250-01).

(2) Generation of Stable CHO FlpIn Cell Lines

CHO FlpIn host cells were transfected with a 9:1 ratio ofpOG44:pcDNA5/FRT expression plasmid DNA using lipofectamine 2000according to the manufacturer's instructions (Invitrogen, Catalog No.11668-027). As controls, cells were transfected with empty pcDNA5/FRTvector/pOG44 and pOG44 plasmid (Invitrogen, Catalog No. 35-3018) alone.Twenty four hours after transfection, the cells were split, and afterforty eight hours 0.5 mg/mL Hygromycin B (Sigma, Catalog No. H0654-SPEC)was added to the cells. Polyclonal selection of stable cells wasperformed in F12K, 10% FCS, 1% Penicillin/Streptomycin, 0.5 mg/mLHygromycin B.

(3) Protein Expression in Stable CHO FlpIn Cell Lines

Approximately 2×10⁶ cells were seeded in 15 cm plates and grown in F12K(ATCC, Catalog No. 30-2004)/DMEM high glucose (Invitrogen, Catalog No.11995065) 1:1, 5% ultra low IgG FCS (Invitrogen, #16250-78) at 37° C.,5% CO₂ for 5-6 days. Supernatants were harvested and resulting proteinsanalyzed by ELISA and by surface plasmon resonance.

Example 4 Binding Characteristics of Anti-hHGF Monoclonal Antibodies

The monoclonal antibodies produced in Example 1 were characterized bytheir ability to bind hHGF, and certain of the recombinant HGF proteinsproduced in Example 3.

The antibodies were analyzed by surface-plasmon resonance using aBIAcore T100 instrument to assess their ability to bind HGF and certainof the fusion proteins discussed in Example 3. Each antibody wasimmobilized on a carboxymethylated dextran CM5 sensor chip (BIAcore,Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No.BR-1000-50) using a standard coupling protocol according tomanufacturer's instructions.

Analyses were performed at 25° C. using PBS (GIBCO, Catalog No.14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No.R-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/mL CM-DextranSodium salt (Fluka, Catalog No. 86524) as running buffer. Supernatantcontaining different HGF fusion proteins or supernatant from cellstransfected with empty vector were injected over each antibody at a flowrate of 30 μL/min for 3 minutes. The resulting binding was determined asresonance units (RU) over baseline 30 seconds after the end ofinjection. Binding was compared to human HGF (R&D Systems, Catalog No.294-HGN-025) diluted in running buffer. Non-specific binding wasmonitored by comparing binding to a control surface where mouse IgG(Rockland, Catalog No. 010-0102) was immobilized using the same aminecoupling procedure.

The results are summarized in the Table 3.

TABLE 3 rhHGF rmHGF mhm (R&D (R&D chimera Sys- Sys- (V495- humanAntibody tems) tems) L585) HGF G555E C561R 1A3 Yes No No Yes Yes Yes 1D3Yes No Yes Yes Yes Yes 1F3 Yes Yes Yes Yes Yes Yes 2B8 Yes No Yes YesYes Yes 2F8 Yes Yes No Yes Yes Yes 3A12 Yes No No Yes Yes Yes 3B6 Yes NoNo Yes Yes Yes 3D11 Yes No No Yes Yes Yes

The results in Table 3 demonstrate that each of the antibodies bind rHGFand purified human HGF. Furthermore, all of the antibodies bind hHGFcontaining point mutations G555E and C561R. In general, all of theantibodies except for 1F3 and 2F8 did not bind murine HGF demonstratingthat the antibodies 1A3, 1D3, 2B8, 3A12, 3B6, and 3D11 specifically bindhuman HGF. Antibodies 1D3, 1F3, and 2B8 bind the mouse-human-mousechimera whereas the remaining antibodies did not. The results suggestthat the antibodies 1D3 and 2B8 at least in part bind to residues495-585 of human HGF. The antibodies 1A3, 3A12, 3B6, and 3D11 appear tobind portions of human hHGF other than residues 495-585. At present, itis uncertain why 2F8 does not bind the mhm chimera as it appears to bindboth hHGF and mHGF.

Example 5 Ability of Anti-hHGF Monoclonal Antibodies to Bind Reduced andNon-Reduced HGF

In this Example, the anti-hHGF monoclonal antibodies produced in Example1 were analyzed for their ability to bind reduced and non-reduced HGF.

The reactivity of the anti-HGF sera with the recombinant hHGF wasassessed by immunoblotting. Eight μg of recombinant hHGF protein inNuPAGE MOPS SDS running buffer (Invitrogen) with or without NuPAGEsample reducing buffer (Invitrogen) was fractionated on a 4-12% Bis-Tris1.0 mm×2D well gel (Invitrogen, Carlsbad, Calif.). The fractionatedproteins then were transferred onto a nitrocellulose membrane usingstandard procedures. The nitrocellulose membranes were blocked with 5%nonfat milk powder solution in Tris buffered Saline with 0.1% Tween-20®(TBST), and then mounted onto a Mini Protean II Multi-Screen apparatus(BioRad) for further blocking.

The resulting membranes were probed with the purified antibodies on aMulti-Screen apparatus. The purified antibodies were diluted to 5 μg/mLin blocking buffer. The nitrocellulose membrane then was removed fromthe apparatus, and incubated with horseradish peroxidase-labeledanti-mouse IgG antibodies. The results are summarized in Table 4, wherethe numbers reflect the extent of binding with − representing the least(little or no binding) and 3+ representing the most binding.

TABLE 4 Reduced Non-Reduced Antibody (exposure: 3-5 min) (exposure: 20sec) 1A3 2+ 2+ 1D3 2+ 2+ 1F3 2+ 2+ 2B8 − 1+ 2F8 2+ 2+ 3A12 − 2+ 3B6 3+2+ 3D11 − 3+

The data in Table 4 demonstrate that all the antibodies bind non-reducedrhHGF. In contrast, monoclonal antibodies 1A3, 1D3, 1F3, 2F8, 3B6 boundreduced rhHGF but antibodies 2B8, 3A12, and 3D11 did not bind to reducedrhHGF.

Example 6 Binding Affinities

The binding affinities and kinetics of interaction of each of theantibodies produced in Example 1 against hHGF were measured by surfaceplasmon resonance.

Rabbit anti-mouse immunoglobulins (BIAcore, Catalog No. BR-1005-14) wereimmobilized on carboxymethylated dextran CM5 sensor chips (BIAcore,Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No.BR-1000-50) using a standard coupling protocol according tomanufacturer's instructions. The analyses were performed at 25° C. usingPBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20(BIAcore, Catalog No. BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930),and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) asrunning buffer.

The antibodies were captured in an individual flow cell at a flow rateof 10 μL/min. Injection time was variable for each antibody to yieldapproximately 20 RU of antibody captured for each cycle. Buffer or HGF(R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer wasinjected sequentially over a reference surface (no antibody captured)and the active surface (antibody to be tested) for 2 minutes at 60μL/min. The dissociation phase was monitored for 15 or 90 minutes,depending on concentration. The surface then was regenerated with 10 mMGlycine-HCl, pH 1.7 (BIAcore, Catalog No. BR-1003-54) injected for 3minutes at a flow rate of 60 μL/min before another cycle was initiated.HGF concentrations tested were 0.46 nM to 7.5 nM.

Kinetic parameters were determined using the kinetic function of theBIAevalutation software with reference subtraction. Kinetic parametersfor each antibody, k_(a) (association rate constant), k_(d)(dissociation rate constant) and K_(D) (equilibrium dissociationconstant) are summarized in Table 5.

TABLE 5 Anti- body ka (1/Ms) SE (ka) kd (1/s) SE (kd) K_(D) (pM) SD 1A31.7 × 10⁶ 7.3 × 10⁴ 5.2 × 10⁻⁵ 8.4 × 10⁻⁷ 30.1 5.6 1D3 1.7 × 10⁶ 3.1 ×10⁴ 8.2 × 10⁻⁵ 1.7 × 10⁻⁶ 54.2 27.4 1F3 1.5 × 10⁶ 5.0 × 10⁴ 2.6 × 10⁻⁵6.6 × 10⁻⁷ 18.1 8.2 2B8 1.6 × 10⁶ 2.9 × 10⁴ 2.1 × 10⁻⁵ 1.4 × 10⁻⁷ 13.54.4 3A12 1.6 × 10⁶ 3.7 × 10⁴ 1.6 × 10⁻⁴ 1.6 × 10⁻⁶ 103.0 10.4 3B6 2.0 ×10⁶ 6.5 × 10⁴ 3.9 × 10⁻⁵ 3.2 × 10⁻⁷ 17.0 3.4

The data in Table 5 demonstrate that the antibodies bind hHGF with aK_(D) of about 100 pM or less, about 50 pM or less, or 20 pM or less.

Example 7 Neutralization Activity of Anti-hHGF Antibodies

In this Example, the antibodies produced in Example 1 were characterizedfor their ability to (a) inhibit the binding of hHGF to c-Met, and (b)inhibit HGF stimulated BrdU incorporation in 4 MBr-5 cells.

a. HGF-Met Binding Inhibition Assay (Neutralization Assay)

The antibodies were tested by ELISA for their ability to inhibit hHGFbinding to c-Met.

Specifically, Wallac 96-well DELFIA assay plates (Wallac Inc., CatalogNo. AAAND-0001) were coated with 100 μL of 6.25 μg/mL HGF (R&D Systems,Catalog No. 294-HGN-025) in carbonate coating buffer (15 mM Na₂CO₃ and34 mM NaHCO₃, pH 9.0) for 16 hours at 4° C. The plates then were blockedwith 200 μL of 5% non-fat dry milk in PBS for 1 hour at roomtemperature. The antibodies were prepared in a separate plate by addingincreasing concentrations of the antibodies under investigation(0.033-667 nM, 3-fold-serial dilution) to 2 nM c-Met (R&D Systems,Catalog No. 358-MT/CF) in 5% non-fat dry milk in PBS. 100 μL of sampleper well was transferred to the assay plate and incubated overnight at4° C. The assay plates then were washed 3 times with PBS-0.1% Tween 20,and incubated for 2 hours at room temperature with 100 μL/well of 2μg/mL biotinylated anti-human c-Met antibody (R&D Systems, Catalog No.BAF358) prepared in 5% non-fat dry milk in PBS.

The resulting plates then were washed three times with PBS-0.1% Tween20, and incubated for 1 hour at room temperature with Eu-labeledStreptavidin (Wallac, Catalog No. 1244-360) diluted 1:1000 in DELFIAassay buffer (Wallac, Catalog No. 4002-0010). The resulting plates werewashed 3 times with DELFIA wash solution (Wallac, Catalog No. 4010-0010)and incubated with 100 μL/well DELFIA enhancement solution (Wallac#4001-0010) for 15 minutes at room temperature with agitation.

The plates were read on Victor³V instrument (Perkin Elmer) using theEuropium method. The IC₅₀ values were calculated and are summarized inTable 6.

TABLE 6 Antibody IC₅₀ (nM) SD 1A3 5.65 0.91 1D3 4.43 2.27 1F3 6.57 0.282B8 5.57 1.19 2F8 5.36 0.88 3A12 5.26 2.11 3B6 − − 3D11 5.66 2.75

The results demonstrate that all the antibodies (i.e., 1D3, 1A3, 2B8,3A12, 1F3, 3D11, and 2F8) other than 3B6 efficiently neutralize HGFbinding to c-Met.

b. Neutralization of HGF Stimulated BrdU Incorporation in 4 MBr-5 Cells

Ten μL of 12.5 nM of hHGF was dispensed into individual wells of a96-well tissue culture microtiter plate (Costar Catalog No. 3903). TenμL of serially diluted antibodies at concentrations of 6667, 2222, 740,247, 82, 27, 9.1, 3.0, 1.0, 0.33 nM were added to each well. The HGFantibody mixture then was incubated at room temperature for 30 minutes.Monkey bronchial epithelial cells 4 MBr-5 (ATCC, CCL208) cultured inF-12K (ATCC, 30-2004), 15% FBS (Gibco 10438-026), 30 ng/mL EGF (SigmaE9644), 1% penicillin/streptomycin (PS, Gibco Catalog No. 15140-122)were dissociated with Trypsin (Gibco Catalog No. 25200-056), resuspendedin assay media (F-12K, 2.5% FBS, 1% PS) at 75,000 cells/mL, and 80 μL ofthe cell suspension was dispensed to the HGF antibody mixture.

The resulting cells were incubated at 37° C., 5% CO₂. Forty eight hourslater, 10 μL of 100 μM BrdU (Roche Catalog No. 1669915) was added.Seventy two hours later, the media was removed, the plates were driedwith a hair dryer and were processed with the BrdU ELISA in accordancewith manufacturer's instructions (Roche Catalog No. 1669915).

The luminescent signal was quantified by a Synergy HT plate reader(Bio-Tek). The data were fit to a sigmoidal dose response with variableslope with the equation y=bottom+(top-bottom)/(1+10^(log(EC50−x)*hillslope)) in GraphPad Prism (GraphPad Software). Each experiment wasrepeated at least 3 times in duplicates, and average EC₅₀ values arepresented in Table 7.

TABLE 7 Antibody IC₅₀ (nM) 1A3 4.69 1D3 4.99 1F3 1.94 2B8 1.41 2F8 19.243A12 30.30 3B6 36.08 3D11 51.12

The results in Table 7 demonstrate that all of the antibodies, 1A3, 1D3,1F3, 2B8, 2F8, 3A12, 3B6, and 3D11 inhibit HGF induced proliferation in4 MBr-5 cells.

Example 8 Anti-Scatter Activity of Anti-hHGF Antibodies

This Example describes a characterization of the antibodies produced inExample 1 for their ability to inhibit HGF induced scatter activity. HGFinduces “scattering” (motility) of clusters in MDCK cells (ATCC,Manassas, Va., Catalog No. CCL-34).

MDCK cells were seeded in 96-well Costar tissue culture plates (CorningIncorporated, Corning, N.Y., Catalog No. 3595) at a density of 4×10³cells per well in 80 μl, MEM (ATCC, Manassas, Va., Catalog No. 30-2003)containing 10% Fetal Bovine Serum (Invitrogen Catalog No. 10438026), and1% penicillin-streptomycin (Invitrogen Catalog No. 15140122). Each ofthe antibodies to be investigated was diluted to 6,667 nM in MEMcontaining 10% Fetal Bovine Serum and 1% penicillin-streptomycin. Eachof the different antibody dilutions, as well as MEM containing 10% FetalBovine Serum and 1% penicillin-streptomycin without antibody, then wasseparately combined with an equal volume of MEM containing 10% FetalBovine Serum and 1% penicillin-streptomycin, and 100 ng/ml HGF (R&DSystems Catalog No. 294-HGN-025). The antibody/HGF dilutions wereincubated for 30 minutes at 25° C. Twenty μL of each antibody/HGFdilution was added separately to individual wells, yielding a finalantibody concentration of 666.7 nM, and a final HGF concentration of 10ng/ml. The MDCK cells then were incubated for 24 hours at 37° C. with 5%CO₂.

After 24 hours incubation, the MDCK cells were carefully washed oncewith 100 μL per well of ice-cold PBS (Invitrogen Catalog No. 14190144),and fixed with 100 μL per well of ice-cold methanol while rocking for 10minutes at 25° C. The plates then were washed carefully once withdistilled water. A volume of 100 μL crystal violet solution, consistingof 0.5% crystal violet (Sigma, St. Louis, Mo., Catalog No. C3886) and50% ethanol in distilled water, was added to each well, and the cellswere incubated for 20 minutes at 25° C. while rocking.

Following staining with crystal violet solution, the cells were washedcarefully three times with distilled water. Then, PBS was added to eachwell to prevent drying of samples. The cells were imaged using the LeicaDMIRB microscope (Leica Microsystems GmbH, Wetzler, Germany), DC500camera (Leica Microsystems GmbH, Wetzler, Germany), and MagnaFire 2.1 Csoftware (Optronics, Goleta, Calif.), and samples were rated for levelof scattering. The results are summarized in Table 8.

TABLE 8 Inhibition of HGF-induced MDCK Cell Scattering Antibody Trial 1Trial 2 1A3 ++ + 1D3 ++ ++ 1F3 + + 2B8 +++ +++ 2F8 + + 3A12 − −/+ 3B6 ++++ 3D11 − − − No Inhibition +++ Very strong, nearly complete inhibition++ Strong inhibition + Detectable inhibition

The results in Table 8 demonstrate that antibody 2B8 inhibitedHGF-induced scattering more than the other antibodies. Antibodies 1D3and 3B6 displayed an intermediate level of inhibition; antibody 1A3displayed a low to intermediate level of inhibition: antibodies 1F3 and2F8 displayed a low level of inhibition; and antibodies 3A12 and 3D11gave little or no detectable inhibition.

Example 9 Inhibition of HGF-Stimulated c-Met Phosphorylation

This Example describes a characterization of the antibodies produced inExample 1 for their ability to inhibit the HGF-stimulated c-Metphosphorylation in PC-3 cells. HGF induces phosphorylation of Met inPC-3 cells (ATCC No. CRL-1435).

PC-3 cells were seeded into individual wells of 96-well Costar tissueculture plates (Corning Catalog No. 3595) at a density of 4.5×10⁴ cellsper well in 100 μL F-12K (ATCC, Manassas, Va., Catalog No. 30-2004)containing 10% Fetal Bovine Serum (Invitrogen Catalog No. 10438026) and1% penicillin-streptomycin (Invitrogen Catalog No. 15140122). After 24hours at 37° C. with 5% CO₂, the media was removed, and cells wererinsed once with serum-free F-12K containing 1% penicillin-streptomycin.Cells then were incubated for 24 hours in 100 μL serum-free F-12Kcontaining 1% penicillin-streptomycin.

The following 10 different dilutions of each of the antibodies beinginvestigated were prepared in serum-free F-12K containing 1%penicillin-streptomycin: 6667 nM, 2222 nM, 741 nM, 247 nM, 82.3 nM, 27.4nM, 9.1 nM, 3.0 nM, 1.0 nM, and 0.3 nM. Each antibody dilution, and,serum-free F-12K containing 1% penicillin-streptomycin without antibody,were separately combined with an equal volume of serum-free F-12Kcontaining 1% penicillin-streptomycin and 500 ng/mL HGF (R&D SystemsCatalog No. 294-HGN-025). These antibody/HGF dilutions were incubatedfor 30 minutes at 25° C. This resulted in a final concentration of 1.25nM HGF.

The PC-3 cells then were rinsed once with serum-free F-12K containing 1%penicillin-streptomycin. Next, 70 μL of serum-free F-12K containing 1%penicillin-streptomycin was added to the cells, followed by 10 μL of 10mM Na₃VO₄ (Sigma Catalog No. S6508) in serum-free F-12K containing 1%penicillin-streptomycin. The cells then were incubated for 60 minutes at37° C. with 5% CO₂. Following this incubation, 20 μL of eachantibody/HGF dilution was added separately to separate wells, yielding afinal HGF concentration of 50 ng/mL, and the following finalconcentrations of each antibody: 666.7 nM, 222.2 nM, 74.1 nM, 24.7 nM,8.23 nM, 2.74 nM, 0.91 nM, 0.30 nM, 0.10 nM, 0.03 nM. The cells thenwere incubated for 10 minutes at 37° C. with 5% CO₂, after which pointthe media/antibody/HGF mixture was removed, the plates were placed onice. The cells then were rinsed once with 100 μL per well of ice-coldPBS (Invitrogen Catalog No. 14190144) containing 1 mM Na₃VO₄. The cellsthen were incubated for 30 minutes at 4° C. in 100 μL per well ice-coldlysis buffer consisting of 1% OmniPur Triton X-100 (MERCK KGaA,Darmstadt, Germany, Catalog No. 9410), 50 mM Tris-HCl pH 8.0, 100 mMNaCl, 0.3 mM Na₃VO₄, 1× protease inhibitor cocktail (Sigma Catalog No.P8340), and 1× phosphatase inhibitor cocktail 2 (Sigma Catalog No.5726).

Biotinylated anti-human HGF-R (c-met) antibody (R&D Systems Catalog No.BAF358) was diluted to a concentration of 2 μg/mL in DELFIA Assay Buffer(PerkinElmer, Turku, Finland, Catalog No. 4002-0010) containing 1%bovine serum albumin (Sigma Catalog No. A2153), and 50 μL of thisdilution was added per well of yellow streptavidin microtitration plates(PerkinElmer Catalog No. AAAND-0005). The plates then were incubatedwith antibody for 30 minutes at 25° C. with rocking. Followingincubation, the plates were washed with DELFIA wash solution(PerkinElmer Catalog No. 4010-0010), and 80 μL of each of the differentPC-3 cell lysates was added separately to individual wells of the washedstreptavidin microtitration plates.

The streptavidin microtitration plates containing PC-3 cell lysates wereincubated for 60 minutes at 25° C. with shaking, and then washed withDELFIA wash solution. 100 μL, of 600 ng/mL DELFIA Eu-N1 P-Tyr-100antibody (PerkinElmer Catalog No. AD0159) diluted in DELFIA Assay Buffercontaining 1% bovine serum albumin was added to each well of the washedstreptavidin microtitration plates previously incubated with PC-3 celllysates. The plates were incubated for 60 minutes at 25° C., withrocking. The plates were washed a final time with DELFIA wash solution.Then 200 μL of DELFIA Enhancement Solution (PerkinElmer Catalog No.4001-0010) was added to each well of the washed streptavidinmicrotitration plates, and the plates were incubated in the dark for 5minutes at 25° C., with shaking.

Signal then was measured using the Europium protocol on the Victor3Vreader (PerkinElmer). EC₅₀ values were calculated using Prism 4 forWindows (GraphPad Software, Inc., San Diego, Calif.) and the sigmoidaldose-response equation.

The results summarized as EC50s in nM are tabulated in Table 9.

TABLE 9 Average of Standard Antibody Two Trials Deviation 1A3 0.6840.242 1D3 0.984 0.129 1F3 1.19 1.01 2B8 0.287 0.104 2F8 1.39 2.12 3A122.00 0.553 3B6 1.01 1.11 3D11 2.28 N/A

The data in Table 9 demonstrate that all eight antibodies are potentinhibitors of HGF-induced c-Met phosphorylation in PC-3 cells.

Example 10 Tumor Inhibition in U87MG Xenograft Model

The ability of murine monoclonal antibodies of the invention to inhibittumor growth was tested in an U87MG xenograft model. U87MG cells (ATCC)were expanded in culture at 37° C. in an atmosphere containing 5% CO2and 95% air, using a medium comprising Dulbecco's Modified Eagle medium(DMEM) with 10% fetal bovine serum, 100 units/mL penicillin and 100μg/mL streptomycin. The cells were subcultured and maintained bydetaching the cells from the wall of the culture dish usingtrypsin-EDTA.

Near-confluent cells were collected by trypsinization and then 5×10⁶cells in 50% Matrigel (BD Biosciences; catalog no. 356237) were injectedsubcutaneously into the upper dorsal area between the shoulder blades of7-week old female ICR SCID mice (Taconic Labs). The long (L) and short(W) diameters (mm) of tumors were measured with a caliper. Tumor volume(vol.) was calculated as: volume (mm³)=L×W²/2. When the tumors grew toapproximately 200 mm³, the tumor-bearing mice were randomized into 5groups of 10 mice each. One group received PBS. Each of the other 4groups received one of the antibody 1A3, 1D3, 1F3 or 2B8. All antibodieswere dosed at 1 mg/kg body weight, twice per week, by intra-peritonealinjections of 5 doses. Tumor volumes and mouse body weights wererecorded twice per week. Tumor growth inhibition was analyzed usingStudent's t-test. The results are summarized in FIG. 6 and Table 10.

TABLE 10 Percent Inhibition 2B8 vs PBS 93% p = 0.001 1A3 vs PBS 73% p =0.0075 1D3 vs PBS 51% p = 0.075 1F3 vs PBS 60% p = 0.027

Partial regression was achieved in 2B8 treated group (FIG. 6).Statistically significant growth inhibition was observed in the1A3-treated and 1F3-treated groups (Table 10). There was 51% tumorgrowth inhibition for 1 D3 with a p value of 0.075. No significant bodyweight loss was observed.

Example 11 Tumor Inhibition in U118 Xenograft Model

The ability of the antibodies 1A3, 1D3, 1F3 and 2B8 to inhibit tumorgrowth was tested in an U118 xenograft model. U118 cells (ATCC) wereexpanded as described in Example 10 (above) with respect to the U87MGcells.

Subcutaneous tumors were established as described in Example 10 above,except that the mice used were 7 weeks old female NCr nude mice(Taconic), and treatment was started when the tumors grew toapproximately 80 mm³. As in the U87MG model, all the antibodies weredosed at 1 mg/kg body weight twice a week by intra-peritoneal injectionsfor 4 doses. Tumor volumes and body weights of the mice were recordedtwice per week. Tumor growth inhibition was analyzed using Student'st-test. The results are summarized in FIG. 7 and Table 11.

TABLE 11 Percent Inhibition 2B8 vs IgG 75% p = 0.007 1A3 vs IgG 57% p =0.01 1D3 vs IgG 47% p = 0.12 1F3 vs IgG 30% p = 0.39

Statistically significant tumor growth inhibition was observed in 2B8and 1A3 treated groups (FIG. 7). There was modest tumor growthinhibition in 1F3 and 1D3 groups with p values less than 0.05, which wasdefined as statistical significance in this study (Table 11). Nosignificant body weight loss was observed.

Example 12 Humanization of Murine Monoclonal Antibodies

This Example describes the humanization of the murine 2B8 antibody,together with a characterization of the resulting humanized antibodies.The murine 2B8 Heavy and Light Variable Regions were “humanized” by twomethods.

A. Humanization Procedure 1

In the first method, three humanized heavy chain variable regions andtwo humanized kappa light chain variable regions were designed based onthe “superhumanization” method described in Hwang et al. (2005) METHODS36:35-42; Tan et al. (2002) J. IMMUNOL. 169:1119-1125; U.S. Pat. No.6,881,557.

The Chothia canonical structural class was determined for each mouse 2B8CDR based on CDR length and amino acid composition. Human germlinevariable regions consisting of the same Chothia canonical structuralclass light and heavy variable regions were identified based on knownhuman germline variable region reference alleles described at theInternational Immunogentics Information System (IMGT) website (availableon the world wide web at imgt.cines.fr andbiochem.unizh.ch/antibody/Sequences/index.html). These human germlinevariable regions of the same structural class were compared to murine2B8 variable regions by calculating the percent identity or similaritybetween CDR amino acid residues. Those human germline variable regionswith the highest identity and/or similarity with mouse 2B8 CDR residueswere chosen for CDR grafting. The framework residues of the humangermline variable regions were preserved while the mouse 2B8 CDRresidues were used to replace the corresponding human germline variableregion residues that were different between mouse 2B8 CDR and humangermline CDRs. The human J region that was most similar to the 2B8 mouseJ region was then added to the carboxyl terminus of the “superhumanized”variable region. A signal sequence was then added to the amino terminusof the “superhumanized” variable regions and these amino acid sequenceswere converted into nucleic acid sequences.

The complete variable region nucleic acid sequence was constructed usinggene synthesis PCR methods (Young et al. (2004) NUCL. ACIDS RES. 32:e59)and cloned into a mammalian expression vector (based on pcDNA3.2 DEST(Invitrogen)) containing human constant IgG1 (G1m(17,1) allotype) orKappa (Km(3) allotype (allele 2)) regions (downstream of the variableregions) using standard molecular biology techniques. All four heavychain IgG1 antibodies (chimeric 2B8 and 3 humanized heavy chains (Hu2B8Hv14.1, Hu2B8 Hv5-a.1, Hu2B8 Hv5-51.1) were expressed in the possiblecombinations with all 3 kappa chain antibodies (chimera 2B8 and 2humanized light chains (Hu2B8 Kv1-39.1 and Hu2B8 Kv3-15.1) creating 12different antibody proteins. Binding of the chimeric,chimeric/humanized, and humanized antibodies to human HGF was thenmeasured as described below and the results are summarized in FIG. 8.Each of the possible combinations of immunoglobulin heavy chain andimmunoglobulin light chain variable regions are set forth below in Table12A.

TABLE 12A Heavy Chain Variable Region Light Chain Variable RegionChimeric 2B8 (SEQ ID NO: 12) Chimeric 2B8 (SEQ ID NO: 14) Chimeric 2B8(SEQ ID NO: 12) Hu2B8 Kv1-39.1 (SEQ ID NO: 173) Chimeric 2B8 (SEQ ID NO:12) Hu2B8 Kv3-15.1 (SEQ ID NO: 179) Hu2B8 Hv1-f.1 (SEQ ID NO: 159)Chimeric 2B8 (SEQ ID NO: 14) Hu2B8 Hv1-f.1 (SEQ ID NO: 159) Hu2B8Kv1-39.1 (SEQ ID NO: 173) Hu2B8 Hv1-f.1 (SEQ ID NO: 159) Hu2B8 Kv3-15.1(SEQ ID NO: 179) Hu2B8 Hv5-a.1 (SEQ ID NO: 165) Chimeric 2B8 (SEQ ID NO:14) Hu2B8 Hv5-a.1 (SEQ ID NO: 165) Hu2B8 Kv1-39.1 (SEQ ID NO: 173) Hu2B8Hv5-a.1 (SEQ ID NO: 165) Hu2B8 Kv3-15.1 (SEQ ID NO: 179) Hu2B8 Hv5-51.1(SEQ ID NO: 169) Chimeric 2B8 (SEQ ID NO: 14) Hu2B8 Hv5-51.1 (SEQ ID NO:169) Hu2B8 Kv1-39.1 (SEQ ID NO: 173) Hu2B8 Hv5-51.1 (SEQ ID NO: 169)Hu2B8 Kv3-15.1 (SEQ ID NO: 179)

Each of the possible combinations of immunoglobulin heavy chains andimmunoglobulin light chains are set forth below in Table 12B.

TABLE 12B Immunoglobulin Heavy Chain Immunoglobulin Light Chain Chimeric2B8 IgG1 (SEQ ID NO: 155) Chimeric 2B8 Kappa (Km(3)) (SEQ ID NO: 157)Chimeric 2B8 IgG1 (SEQ ID NO: 155) Hu2B8 Kv1-39.1 + Kappa Constant(Km(3) allotype) (allele 2) (SEQ ID NO: 177) Chimeric 2B8 IgG1 (SEQ IDNO: 155) Hu2B8 Kv3-15.1 + Kappa Constant (Km(3) allotype) (allele 2)(SEQ ID NO: 181) Hu2B8 Hv1-f.1 + IgG1 Constant Chimeric 2B8 Kappa(Km(3)) (G1M(17,1)) allotype (SEQ ID NO: (SEQ ID NO: 157) 163) Hu2B8Hv1-f.1 + IgG1 Constant Hu2B8 Kv1-39.1 + Kappa Constant (G1M(17,1))allotype (SEQ ID NO: (Km(3) allotype) (allele 2) (SEQ ID 163) NO: 177)Hu2B8 Hv1-f.1 + IgG1 Constant Hu2B8 Kv3-15.1 + Kappa Constant(G1M(17,1)) allotype (SEQ ID NO: (Km(3) allotype) (allele 2) (SEQ ID163) NO: 181) Hu2B8 Hv5-a.1 + IgG1 Constant Chimeric 2B8 Kappa (Km(3))(G1M(17,1)) allotype (SEQ ID NO: (SEQ ID NO: 157) 167) Hu2B8 Hv5-a.1 +IgG1 Constant Hu2B8 Kv1-39.1 + Kappa Constant (G1M(17,1)) allotype (SEQID NO: (Km(3) allotype) (allele 2) (SEQ ID 167) NO: 177) Hu2B8 Hv5-a.1 +IgG1 Constant Hu2B8 Kv3-15.1 + Kappa Constant (G1M(17,1)) allotype (SEQID NO: (Km(3) allotype) (allele 2) (SEQ ID 167) NO: 181) Hu2B8Hv5-51.1 + IgG1 Constant Chimeric 2B8 Kappa (Km(3)) (G1M(17,1)) allotype(SEQ ID NO: (SEQ ID NO: 157) 171) Hu2B8 Hv5-51.1 + IgG1 Constant Hu2B8Kv1-39.1 + Kappa Constant (G1M(17,1)) allotype (SEQ ID NO: (Km(3)allotype) (allele 2) (SEQ ID 171) NO: 177) Hu2B8 Hv5-51.1 + IgG1Constant Hu2B8 Kv3-15.1 + Kappa Constant (G1M(17,1)) allotype (SEQ IDNO: (Km(3) allotype) (allele 2) (SEQ ID 171) NO: 181)

Two of the possible antibody constructs containing the full lengthimmunoglobulin heavy and light chains containing humanized variableregions are designated below:

-   -   sh2B8-9 (G1m(17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region        (G1m(17,1) allotype) (SEQ ID NO. 171) plus hu2B8 Kv 1-39.1        (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID NO.        177)    -   sh2B8-12 (G1m(17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region        (G1m(17,1) allotype)) (SEQ ID NO. 171) plus hu2B8 Kv 3-15.1        (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID NO.        181).

The nucleic acid sequences encoding and the protein sequences definingeach of the humanized antibodies are summarized below. In this section,the last nucleotide of each variable region is the first base of thenext codon generated by the variable/constant region junction. Thisnucleotide is included in the Variable Region because it is part of thatexon. Amino acid sequences of Constant Regions listed below include thetranslation of this junction codon.

(1) Nucleic Acid Sequence Encoding the Full Length Chimeric 2B8 HeavyChain (Mouse Variable Region and Human IgG1 Constant Region) (AllotypeG1m(17,1)) (Signal Sequence Underlined) (SEQ ID NO. 154)

   1 atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag  61 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc 121 tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct 181 ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat 241 gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 301 caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt 361 ggtagcatct ttgactactg gggccaaggc accactctca ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381 ctctccctgt ctccgggtaa atga

(2) Protein Sequence Defining the Full Length Chimeric 2B8 Heavy Chain(Chimeric 2B8 IgG1 (G1m(17,1) Allotype) (without Signal Sequence) (SEQID NO. 155)

  1 qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny 61 nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gttltvssas121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421 gnvfscsvmh ealhnhytqk slslspgk

(3) Nucleic Acid Sequence Encoding the Full Length Chimeric 2B8 LightChain (Mouse Variable Region and Human Constant Region) (Chimeric 2B8Kappa (Km(3))) (Signal Sequence Underlined) (SEQ ID NO. 156)

  1 atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg 61 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc121 ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca181 gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat241 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct301 gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg361 gggaccaggc tggaaataaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca421 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga

(4) Protein Sequence Defining the Full Length Chimeric 2B8 Light Chain(Chimeric 2B8 Kappa (Km(3))) (without Signal Sequence) (SEQ ID NO. 157)

  1 nivmtqspks msmsvgervt lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd 61 rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrtv aapsvfifpp121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt181 lskadyekhk vyacevthqg lsspvtksfn rgec

(5) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv1-f.1 Heavy ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 158)

  1 atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag 61 gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc121 tgcaaggttt ctggatacac cttcaccacc tactggatgc actgggtgca acaggcccct181 ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat241 gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg301 gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag

(6) Protein Sequence Defining Humanized Hu2B8 Hv1-f.1 Heavy ChainVariable Region (without Signal Sequence) (SEQ ID NO. 159)

 1 evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny 61nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvss

(7) Nucleic Acid Sequence Encoding Human IgG1 Heavy Chain ConstantRegion (G1m(17,1) Allotype) (SEQ ID NO. 160)

  1 cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag181 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct241 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttgagccca301 aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac361 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt481 acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca541 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg601 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca661 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc721 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc841 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc901 agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc961 agaagagcct ctccctgtct ccgggtaaat ga

(8) Protein Sequence Defining Human IgG1 Heavy Chain Constant Region(G1m(17,1) Allotype) (SEQ ID NO. 161).

The first Amino acid is derived from translation of the last nucleotideof variable region and beginning two nucleotides of the IgG1 Heavy Chainsequence.

  1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep kscdkthtcp pcpapellgg121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsrde241 ltknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw301 qqgnvfscsv mhealhnhyt qkslslspgk

(9) Nucleic Acid Sequence Encoding the Full Length Heavy Chain HumanizedHu2B8 Hv1f.1 Variable Region and Human IgG1 (G1m(17,1) Allotype) HeavyChain Constant Region (Signal Sequence Underlined) (SEQ ID NO. 162)

   1 atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag  61 gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc 121 tgcaaggttt ctggatacac cttcaccacc tactggatgc actgggtgca acaggcccct 181 ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat 241 gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg 301 gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt 361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381 ctctccctgt ctccgggtaa atga

(10) Protein Sequence Defining the Full Length Heavy Chain HumanizedHu2B8 Hv1f.1 Variable Region and Human IgG1 Heavy Chain Constant Region(G1m(17,1) Allotype) (Without Signal Sequence) (SEQ ID NO. 163)

  1 evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny 61 nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvssas121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421 gnvfscsvmh ealhnhytqk slslspgk

(11) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv5a.1 Heavy ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 164)

  1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat241 ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag

(12) Protein Sequence Defining Humanized Hu2B8 Hv5a.1 Heavy ChainVariable Region (without Signal Sequence) (SEQ ID NO. 165)

 1 evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny 61npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss

(13) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8Hv5a.1 Heavy Chain Variable Region and Human IgG1 (G1m(17,1) Allotype)Heavy Chain Constant Region (Signal Sequence underlined) (SEQ ID NO.166)

   1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa  61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc 121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 241 ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg 301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381 ctctccctgt ctccgggtaa atga

(14) Protein Sequence Defining the Full Length Humanized Hu2B8 Hv5a.1Heavy Chain Variable Region and Human IgG1 (G1m(17,1) Allotype) HeavyChain Constant Region (without Signal Sequence) (SEQ ID NO. 167)

  1 evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny 61 npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421 gnvfscsvmh ealhnhytqk slslspgk

(15) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv5-51.1 Heavy ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 168)

  1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa 61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat241 ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag

(16) Protein Sequence Defining Humanized Hu2B8 Hv5-51.1 Heavy ChainVariable Sequence (without Signal Sequence) (SEQ ID NO. 169)

 1 evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny 61npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss

(17) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8Hv5-51.1 Heavy Chain Variable Region and Human IgG1 (G1m(17,1) Allotype)Heavy Chain Constant Region (Signal Sequence Underlined) (SEQ ID NO.170)

   1 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa  61 gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 121 tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 181 gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 241 ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg 301 cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 361 ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 721 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381 ctctccctgt ctccgggtaa atga

(18) Protein Sequence Defining the Full Length Humanized Hu2B8 Hv5-51.1Heavy Chain Variable Region and Human IgG1 (G1m(17,1) Allotype) HeavyChain Constant Region (without Signal Sequence) (SEQ ID NO. 171)

  1 evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny 61 npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421 gnvfscsvmh ealhnhytqk slslspgk

(19) Nucleic Acid Sequence Encoding Humanized Hu2B8 Kv1-39.1 Kappa ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 172).

Two possible start ATGs are shown in uppercase.

  1 ATGgacATGa gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 61 agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga121 gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag181 aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc241 ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg301 caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt361 ggccagggga ccaagctgga gatcaaac

(20) Protein Sequence Defining Humanized Hu2B8 Kv1-39.1 Kappa ChainVariable Region (without Signal Sequence) (SEQ ID NO. 173)

 1 diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps 61rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleik

(21) Nucleic Acid Sequence Encoding Human Kappa Chain Constant Region(Km(3) Allotype) (Allele 2) (SEQ ID NO. 174)

  1 gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 61 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt121 ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca181 gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga241 aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga301 gcttcaacag gggagagtgt tga

(22) Protein Sequence Defining Human Kappa Chain Constant Region (Km(3)Allotype) (Allele 2) (SEQ ID NO. 175).

The first amino acid is derived from translation of the last nucleotideof variable region and beginning two nucleotides of the Kappa LightChain sequence.

 1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd 61skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

(23) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8Kv1-39.1 Light Chain Variable Region and Human Kappa Chain ConstantRegion (Km(3) Allotype) (Allele 2) (Signal Sequence Underlined) (SEQ IDNO. 176)

  1 atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 61 agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga121 gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag181 aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc241 ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg301 caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt361 ggccagggga ccaagctgga gatcaaacga actgtggctg caccatctgt cttcatcttc421 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac481 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac541 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc601 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat661 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgttg a

(24) Protein Sequence Defining the Full Length Humanized Hu2B8 Kv1-39.1Light Chain Variable Region and Human Kappa Chain Constant Region (Km(3)Allotype) (Allele 1) (SEQ ID NO. 177)

  1 diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps 61 rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleikrtv aapsvfifpp121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt181 lskadyekhk vyacevthqg lsspvtksfn rgec

(25) Nucleic Acid Sequence Encoding Humanized Hu2B8 Kv3-15.1 Light ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 178)

  1 atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga 61 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc121 ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct181 ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc241 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct301 gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag361 gggaccaagc tggagatcaa ac

(26) Protein Sequence Defining Humanized Hu2B8 Kv3-15.1 Light ChainVariable Region (without Signal Sequence) (SEQ ID NO. 179)

 1 eivmtqspat lsyspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa 61rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleik

(27) Nucleic Acid Encoding the Full Length Humanized Hu2B8 Kv3-15.1Light Chain Variable Region and Human Kappa Chain Constant Region (Km(3)Allotype) (Allele 2) (Signal Sequence Underlined) (SEQ ID NO. 180)

  1 atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga 61 gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc121 ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct181 ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc241 aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct301 gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag361 gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca421 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat481 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag541 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg601 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc661 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga

(28) Protein Sequence Defining Humanized Hu2B8 Kv3-15.1 Light ChainVariable Region and Human Kappa Chain Constant Region (Km(3) Allotype)(Allele 2) (without Signal Sequence) (SEQ ID NO. 181)

  1 eivmtqspat lsyspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa 61 rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleikrtv aapsvfifpp121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt181 lskadyekhk vyacevthqg lsspvtksfn rgec

For convenience, Table 13 provides a concordance chart showing thecorrespondence between the full length sequences and of the antibodiesdiscussed in this section with those presented in the Sequence Listing.

TABLE 13 SEQ. ID NO. Protein or Nucleic Acid 154 Chimeric 2B8 IgG1(Glm(17,1)) - nucleic acid 155 Chimeric 2B8 IgG1 (Glm(17,1)) - protein156 Chimeric 2B8 Kappa (Km(3)) - nucleic acid 157 Chimeric 2B8 Kappa(Km(3)) - protein 158 Hu2B8 Hv1f.1 Heavy Chain Variable Region - nucleicacid 159 Hu2B8 Hv1f.1 Heavy Chain Variable Region - protein 160 HumanIgG1 Heavy Chain Constant Region (G1m(17,1)) allotype - nucleic acid 161Human IgG1 Heavy Chain Constant Region (G1m(17,1)) allotype - protein162 Hu2B8 Hv1f.1 + IgG1 Constant (Glm(17,1) allotype) - nucleic acid 163Hu2B8 Hv1f.1 + IgG1 Constant (Glm(17,1) allotype) - protein 164 Hu2B8Hv5a.1 Heavy Chain Variable Region - nucleic acid 165 Hu2B8 Hv5a.1 HeavyChain Variable Region - protein 166 Hu2B8 Hv5a.1 + IgG1 Constant(Glm(17,1) allotype) - nucleic acid 167 Hu2B8 Hv5a.1 + IgG1 Constant(Glm(17,1) allotype) - protein 168 Hu2B8 Hv5-51.1 Heavy Chain VariableRegion - nucleic acid 169 Hu2B8 Hv5-51.1 Heavy Chain Variable Region -protein 170 Hu2B8 Hv5-51.1 + IgG1 Constant (G1m(17,1 allotype) - nucleicacid 171 Hu2B8 Hv5-51.1 + IgG1 Constant (G1m(17,1 allotype) - protein172 Hu2B8 Kv1-39.1 Kappa Chain Variable Region - nucleic acid 173 Hu2B8Kv1-39.1 Kappa Chain Variable Region - protein 174 Human Kappa ChainConstant Region (Km(3) allotype) (allele 2) - nucleic acid 175 HumanKappa Chain Constant Region (Km(3) allotype) (allele 2) - protein 176Hu2B8 Kv1-39.1 + Kappa Constant (Km(3) allotype) (allele 2) - nucleicacid 177 Hu2B8 Kv1-39.1 + Kappa Constant (Km(3) allotype) (allele 2) -protein 178 Hu2B8 Kv3-15.1 Kappa Chain Variable Region - nucleic acid179 Hu2B8 Kv3-15.1 Kappa Chain Variable Region - protein 180 Hu2B8Kv3-15.1 + Kappa Constant (Km(3) allotype) (allele 2) - nucleic acid 181Hu2B8 Kv3-15.1 + Kappa Constant (Km(3) allotype) (allele 2) - proteinB. Humanization Procedure 2

The second humanization method employed for reducing immunogenicity ofthe mouse 2B8 antibody is based on the method described in Studnicka etal. (1994) PROTEIN ENG. 7:805-814. The heavy and kappa human germlinevariable regions most identical (at the amino acid level) to those ofmouse 2B8 were identified. Residues that differed between mouse andhuman were converted into the human sequence depending on the likelyrisk that such a change would affect binding or immunogenicity. Low riskresidues (i.e., residues that when changed would likely not affectantigen binding and would also reduce potential immunogenicity) werechanged to the human amino acid in the heavy variable region (creatingLR2B8HC) and the kappa variable region (creating LR2B8LC). Additionally,low risk and medium risk (i.e., residues that when changed are somewhatlikely to have an effect on antigen binding residues and would alsoreduce potential immunogenicity) were changed to the human amino acid inthe heavy variable region (creating LRMR2B8HC) and the kappa variableregion (creating LRMR2B8LC). The human IgG1 heavy chain constant region(G1m(3) allotype (allele 1)) was added to the carboxyl terminus of thetwo human engineered heavy variable regions and the human Kappa constantregion (Km(3) allotype (allele 1)) was added to the carboxyl terminus oftwo human engineered light variable regions, thus creating four humanengineered antibody chains. Variable region nucleic acid sequences werefirst synthesized by gene synthesis methods and then added to humanconstant region sequences. These human engineered antibodies were clonedinto mammalian protein expression vectors, and protein was expressed inthe four possible combinations of heavy chain plus light chain. Bindingof the chimeric, chimeric/humanized, or humanized antibodies to humanHGF was measured using conventional techniques, as described below.

The nucleic acid sequences encoding and the protein sequences definingeach of the humanized antibodies are summarized below. In this section,the last nucleotide of each variable region is the first base of thenext codon generated by the variable/constant region junction. Thisnucleotide is included in the Variable Region because it is part of thatexon. Amino acid sequences of Constant Regions listed below include thetranslation of this junction codon.

(1) Nucleic Acid Sequence Encoding the Humanized LR2B8HC Heavy ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 182)

  1 atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa 61 gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca121 tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc181 ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat241 gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg301 gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta361 ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc ag

(2) Protein Sequence Defining Humanized LR2B8HC Heavy Chain VariableRegion (without Signal Sequence) (SEQ ID NO. 183)

 1 qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny 61nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss

(3) Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain ConstantRegion (G1m(3) Allotype) (Allele 1) (SEQ ID NO. 184)

  1 ccagcacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 61 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt121 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag181 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct241 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca301 aatcttgtga caaaactcac acatgtccac cgtgcccagc acctgaactc ctggggggac361 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg421 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt481 acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca541 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg601 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca661 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga721 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg781 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc841 tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag agcaggtggc901 agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc961 agaagagcct ctccctgtcc ccgggtaaat ga

(4) Protein Sequence Defining Human IgG1 Heavy Chain Constant Region(G1m(3) Allotype) (Allele 1 or 2) (SEQ ID NO. 185).

The first amino acid is derived from translation of the last nucleotideof variable region and the beginning two nucleotides of the IgG1 HeavyChain sequence.

  1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg121 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn181 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw301 qqgnvfscsv mhealhnhyt qkslslspgk

(5) Nucleic Acid Sequence Encoding the Full Length Heavy Chain HumanizedLR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy Chain ConstantRegion (G1m(3) Allotype) (Allele 1) (Signal Sequence Underlined) (SEQ IDNO. 186)

   1 atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa  61 gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca 121 tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc 181 ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat 241 gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg 301 gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta 361 ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc agccagcaca 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 721 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381 ctctccctgt ccccgggtaa atga

(6) Protein Sequence Defining the Full Length Heavy Chain HumanizedLR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy Chain ConstantRegion (G1m(3) Allotype) (Allele 1) (without Signal Sequence) (SEQ IDNO. 187)

  1 qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny 61 nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvssas121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181 yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps241 vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301 yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt361 knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421 gnvfscsvmh ealhnhytqk slslspgk

(7) Nucleic Acid Sequence Encoding the Humanized LRMR2B8HC Heavy ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 188)

  1 atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag 61 gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca121 tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc181 ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat241 caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg301 gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta361 ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc ag

(8) Protein Sequence Defining Humanized LRMR2B8HC Heavy Chain VariableRegion (without Signal Sequence) (SEQ ID NO. 189)

 1 qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny 61nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss

(9) Nucleic Acid Sequence Encoding the Full Length Heavy Chain HumanizedLRMR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy ChainConstant Region (G1m(3) Allotype) (Allele 1) (Signal SequenceUnderlined) (SEQ ID NO. 190)

   1 atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag  61 gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca 121 tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc 181 ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat 241 caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg 301 gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta 361 ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc agccagcaca 421 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661 aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 721 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa1081 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag1141 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg1321 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381 ctctccctgt ccccgggtaa atga

(10) Protein Sequence Defining the Full Length Heavy Chain HumanizedLRMR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy ChainConstant Region (G1m(3) Allotype) (Allele 1) (without Signal Sequence)(SEQ ID NO. 191)

  1  qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny 61  nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvssas121  tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181  yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps241  vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301  yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt361  knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421  gnvfscsvmh ealhnhytqk slslspgk

(11) Nucleic Acid Sequence Encoding the Humanized LR2B8LC Light ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 192)

  1  atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc 61  gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc121  cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc181  ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac241  agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca301  gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa

(12) Protein Sequence Defining Humanized LR2B8LC Light Chain VariableRegion (without Signal Sequence) (SEQ ID NO. 193)

 1  divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd61  rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik

(13) Nucleic Acid Sequence Encoding the Human Kappa Chain ConstantRegion (Km(3) Allotype) (Allele 1) (SEQ ID NO. 194)

  1  gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 61  gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt121  ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca181  gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga241  aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga301  gcttcaacag gggagagtgt tag

(14) Protein Sequence Defining the Human Kappa Chain Constant Region(Km(3) Allotype) (Allele 1) (SEQ ID NO. 195).

The first amino acid derived from translation of the last nucleotide ofvariable region and beginning two nucleotides of the Kappa Light Chainsequence.

 1  rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd61  skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

(15) Nucleic Acid Sequence Encoding the Full Length Humanized LR2B8LCLight Chain Variable Region and the Human Kappa Chain Constant Region(Km(3) Allotype) (Allele 1) (SEQ ID NO. 196)

  1  atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc 61  gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc121  cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc181  ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac241  agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca301  gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa361  ggaaccaaac tcgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca421  tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat481  cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag541  gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg601  ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc661  ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag

(16) Protein Sequence Encoding the Full Length Humanized LR2B8LC LightChain Variable Region and the Human Kappa Chain Constant Region (Km(3)Allotype) (Allele 1) (SEQ ID NO. 197)

  1  divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd 61  rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp121  sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt181  lskadyekhk vyacevthqg lsspvtksfn rgec

(17) Nucleic Acid Sequence Encoding the Humanized LRMR2B8LC Light ChainVariable Region (Signal Sequence Underlined) (SEQ ID NO. 198)

  1  atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga 61  gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca121  cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct181  ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac241  agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc301  gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa361  ggcactaaac ttgaaattaa ac

(18) Protein Sequence Defining the Humanized LRMR2B8LC Light ChainVariable Region (without Signal Sequence) (SEQ ID NO. 199)

 1  divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspklliyg asnresgvpd61  rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik

(19) Nucleic Acid Sequence Encoding the Full Length Humanized LRMR2B8LCLight Chain Variable Region and the Human Kappa Chain Constant Region(Km(3) Allotype) (Allele 1) (Signal Sequence Underlined) (SEQ ID NO.200)

  1  atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga 61  gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca121  cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct181  ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac241  agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc301  gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa361  ggcactaaac ttgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca421  tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat481  cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag541  gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg601  ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc661  ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag

(20) Protein Sequence Defining the Full Length Humanized LRMR2B8LC LightChain Variable Region and the Human Kappa Chain Constant Region (Km(3)Allotype) (Allele 1) (SEQ ID NO. 201)

  1  divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspklliyg asnresgvpd 61  rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp121  sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt181  lskadyekhk vyacevthqg lsspvtksfn rgec

For convenience, Table 14 provides a concordance chart showing thecorrespondence between the full length sequences and of the antibodiesdiscussed in this section with those presented in the Sequence Listing.

TABLE 14 SEQ. ID NO. Protein or Nucleic Acid 182 LR2B8HC Heavy ChainVariable Region - nucleic acid 183 LR2B8HC Heavy Chain Variable Region -protein 184 Human IgG1 Heavy Chain Constant Region (G1m(3) allotype)(allele 1) - nucleic acid 185 Human IgG1 Heavy Chain Constant Region(G1m(3) allotype) (allele 1) - protein 186 LR2B8HC + IgG1 Constant(G1m(3) allotype) (allele 1) - nucleic acid 187 LR2B8HC + IgG1 Constant(G1m(3) allotype) (allele 1) - protein 188 LRMR2B8HC Heavy ChainVariable Region - nucleic acid 189 LRMR2B8HC Heavy Chain VariableRegion - protein 190 LRMR2B8HC + IgG1 Constant (G1m(3) allotype)(allele 1) - nucleic acid 191 LRMR2B8HC + IgG1 Constant (G1m(3)allotype) (allele 1) - protein 192 LR2B8LC Light Chain Variable Region -nucleic acid 193 LR2B8LC Light Chain Variable Region - protein 194 HumanKappa Chain Constant Region (Km(3) allotype) (allele 1) - nucleic acid195 Human Kappa Chain Constant Region (Km(3) allotype) (allele 1) -protein 196 LR2B8LC + Kappa Constant (Km(3) allotype) (allele 1) -nucleic acid 197 LR2B8LC + Kappa Constant (Km(3) allotype) (allele 1) -protein 198 LRMR2B8LC Light Chain Variable Region - nucleic acid 199LRMR2B8LC Light Chain Variable Region - protein 200 LRMR2B8LC + KappaConstant (Km(3) allotype) (allele 1) - nucleic acid 201 LRMR2B8LC +Kappa Constant (Km(3) allotype) (allele 1) - protein

Table 15 summarizes the heavy chain CDR sequences (Kabat Definition) ofthe humanized 2B8 antibodies prepared by humanization procedure 1 and byhumanization procedure 2 described herein above in this Example.

TABLE 15 Full Length Heavy Chain Variable Antibody CDR1 CDR2 CDR3 RegionMurine 2B8 TYWMH EINPTNGHTNYNEKFKS NYVGSIFDY SEQ ID NO: Heavy (SEQ IDNO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) 12 Hu2B8 Hv1f.1 TYWMHEINPTNGHTNYNEKFQG NYVGSIFDY SEQ ID NO: (SEQ ID NO: 15) (SEQ ID NO: 202)(SEQ ID NO: 17) 159 Hu2B8 Hv5a.1 TYWMH EINPTNGHTNYNPSFQG NYVGSIFDY SEQID NO: (SEQ ID NO: 15) (SEQ ID NO: 203) (SEQ ID NO: 17) 165 Hu2B8 Hv5-TYWMH EINPTNGHTNYNPSFQG NYVGSIFDY SEQ ID NO: 51.1 (SEQ ID NO: 15) (SEQID NO: 203) (SEQ ID NO: 17) 169 LR2B8HC TYWMH EINPTNGHTNYNEKFKGNYVGSIFDY SEQ ID NO: (SEQ ID NO: 15) (SEQ ID NO: 204) (SEQ ID NO: 17)183 LRMR2B8HC TYWMH EINPTNGHTNYNQKFQG NYVGSIFDY SEQ ID NO: (SEQ ID NO:15) (SEQ ID NO: 205) (SEQ ID NO: 17) 189

Table 16 summarizes the light chain CDR sequences (Kabat Definition) ofthe humanized 2B8 antibodies prepared by humanization procedure 1 and byhumanization procedure 2 described herein above in this Example.

TABLE 16 Full Length Light Chain Variable Antibody CDR1 CDR2 CDR3 RegionMurine 2B8 Light KASENVVSYVS GASNRNT GQSYNYPYT SEQ ID NO: 14 (SEQ ID NO:18) (SEQ ID NO: 19) (SEQ ID NO: 20) Hu2B8 Kv1-39.1 KASENVVSYVS GASNRNTGQSYNYPYT SEQ ID NO: 173 (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20)Hu2B8 Kv3-15.1 KASENVVSYVS GASNRNT GQSYNYPYT SEQ ID NO: 179 (SEQ ID NO:18) (SEQ ID NO: 19) (SEQ ID NO: 20) LR2B8LC KASENVVSYVS GASNRNTGQSYNYPYT SEQ ID NO: 193 (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20)LRMR2B8LC KASENVVSYVS GASNRES GQSYNYPYT SEQ ID NO: 199 (SEQ ID NO: 18)(SEQ ID NO: 206) (SEQ ID NO: 20)C. Binding Affinity of Humanized 2B8 Antibodies

Antigen-binding affinity and kinetics of interaction were assessed bysurface plasmon resonance technology using a BIAcore T100 instrument.Mouse anti-human immunoglobulins (Jackson ImmunoResearch Labs,209-005-098) were immobilized on carboxymethylated dextran CM4 sensorchips (BIAcore, Catalog No. BR-1005-34) by amine coupling (BIAcore,Catalog No. BR-1000-50) using a standard coupling protocol according tomanufacturer's recommendations. The analyses were performed at 25° C.using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20(BIAcore, Catalog No. BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930)and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) asrunning buffer.

The antibodies were captured on individual flow cell at a flow rate of10 μL/min. Injection time was variable for each antibody to yieldapproximately 20 RU of antibody captured for each cycle. Buffer or HGF(R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer wasinjected sequentially over a reference surface (no antibody captured)and the active surface (antibody to be tested) for 2 minutes at 60μL/min. The dissociation phase was monitored for 15 or 90 minutes,depending on concentration. The surface then was regenerated with 10 mMGlycine-HCl, pH 2.0 (BIAcore, Catalog No. BR-1003-55) injected for 3minutes at a flow rate of 60 μL/min before another cycle was initiated.HGF concentrations tested were 1.88, 3.75 and 7.5 nM. Determination ofkinetic parameters was achieved using the kinetic function of theBIAevalutation software with reference subtraction. Kinetic parametersfor each antibody, k_(a) (association rate constant), k_(d)(dissociation rate constant) and K_(D) (equilibrium dissociationconstant) are summarized in FIG. 8.

The results summarized in FIG. 8 show that certain combinations ofsuperhumanized heavy chains (Hu2B8 Hv5a.1, Hu2B8 Hv5-51.1 or Hu2B8Hv1-f.1) and light chains (Hu2B8 Kv1-39.1 or Hu2B8 Kv3-15.1) retainsimilar binding affinity (K_(D)) to HGF as chimeric 2B8 (mouse variableregions with human constant regions) and 2B8 (Table 5).

D. Mutually Exclusive Binding Assay

Mutually exclusive binding to HGF was assessed by surface plasmonresonance technology using a BIAcore T100 instrument. Mouse anti-humanimmunoglobulins (Jackson ImmunoResearch Labs, 209-005-098) wereimmobilized on carboxymethylated dextran CM5 sensor chips (BIAcore,Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No.BR-1000-50) using a standard coupling protocol according tomanufacturer's recommendations. The analyses were performed at 25° C.using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20(BIAcore, #BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/mlCM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running buffer.

The humanized antibodies were captured on an individual flow cell at aflow rate of 30 μL/min. Injection time was variable for each antibody toyield approximately 150 RU of antibody captured for each cycle. HGF (R&DSystems, Catalog No. 294-HGN-025) diluted in running buffer at a finalconcentration of 7.5 μg/mL was injected for 90 sec at 30 μL/min over thecaptured humanized antibodies. Binding of HGF was monitored beforesubsequent injection of mouse 2B8 antibody or polyclonal goat anti-HGFantibody (R & D Systems, AF294) for 3 min at 30 μL/min. The surface thenwas regenerated with 10 mM Glycine-HCl, pH 2.0 (BIAcore, Catalog No.BR-1003-55) injected for 3 min at a flow rate of 60 μL/min beforeanother antibody was tested. The results are summarized in FIG. 9.

Results summarized in FIG. 9 show that both humanized 2B8 antibodies andchimeric 2B8 antibodies prevent murine 2B8 from binding HGF. Theseresults demonstrate that the humanized antibodies still bind the sameHGF epitope as the original 2B8 antibody.

Example 13 Production of Humanized 2B8 Variants

a. HUMAN ENGINEERED™ Antibodies

Codon- and expression-optimized low risk and low-plus-moderate riskHuman Engineered light chain (LR2B8LC and LRMR2B8LC, respectively) andheavy chains (LR2B8HC and LRMR2B8HC, respectively) were cloned in-phaseinto XOMA's transient antibody expression vectors, which contain humanKappa and Gamma-1 constant regions modules. The four Human Engineered2B8 variants were produced by transient transfection in HEK293E cells.The following four antibodies were produced:

-   -   HE2B8-1=LR2B8HC (+IgG1 constant region (G1m(3) allotype (allele        1)) (SEQ ID NO. 187) plus LR2B8LC (+Kappa constant region (Km(3)        allotype (allele 1))) (SEQ ID NO. 197)    -   HE2B8-2=LR2B8HC (+IgG1 constant region (G1m(3) allotype (allele        1)) (SEQ ID NO. 187) plus LRMR2B8LC (+Kappa constant region        (Km(3) allotype (allele 1))) (SEQ ID NO. 201)    -   HE2B8-3=LRMR2B8HC (+IgG1 constant region (G1m(3) allotype        (allele 1)) (SEQ ID NO. 191) plus LR2B8LC (+Kappa constant        region (Km(3) allotype (allele 1))) (SEQ ID NO. 197)    -   HE2B8-4=LRMR2B8HC (+IgG1 constant region (G1m(3) allotype        (allele 1)) (SEQ ID NO. 191) plus LRMR2B8LC (+Kappa constant        region (Km(3) allotype (allele 1))) (SEQ ID NO. 201)

The light and heavy chains were co-transfected into XOMA's suspensionadapted HEK293E cells grown in IS293 media (Irvine Scientific, Irvine,Calif.) using 2 liter shake flasks. After 24 hours in the shake flasks,200 mL of transfected cells were centrifuged, resuspended in 40 mL offresh medium and transferred to Integra flasks (Wilson WolfManufacturing Inc., MN) for production. After incubation for seven days,the cell suspensions were removed from the Integra flasks, centrifugedand the culture supernatants retained. Antibodies in the culturesupernatants were purified on protein A spin columns (Pro-Chem),dialyzed against PBS, concentrated and sterile filtered.

b. SUPERHUMANIZED™ Antibodies

Full length Hu2B8_Hv5-51.1+human IgG1 constant domain (G1m(3) allotype)cDNA was cloned into pEE6.4 (Lonza Biologics, Berkshire, UK) usingHindIII and EcoRI restriction sites. Full length Hu2B8_Kv1-39.1 variableregion+human Kappa constant domain cDNA and full length Hu2B8_Kv3-15.1variable region+human Kappa constant domain cDNA were each cloned intopEE14.4 (Lonza Biologics) using HindIII and EcoRI restriction sites. ThehCMV-MIE promoter+full length Hu2B8_Hv5-51.1+human IgG1 constant domain(G1m(3) allotype) cDNA+SV40 poly A fragment (in pEE6.4) was removed byNotI/SalI digestion and inserted into either Kappa chain pEE14.4 vectorthrough NotI/SalI sites, thus creating 2 different expression vectorsthat each simultaneously express heavy and light chain to make thefollowing antibodies:

-   -   sh2B8-9 (G1m(3))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(3)        allotype) (allele 2)) (SEQ ID NO. 210) plus hu2B8 Kv 1-39.1        (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID NO:        177)    -   sh2B8-12 (G1m(3))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(3)        allotype) (allele 2)) (SEQ ID NO. 210) plus hu2B8 Kv 3-15.1        (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID No.        181)

The nucleic acid sequences encoding and the protein sequences definingthe human IgG1 Heavy Constant Region G1m(3) allotype (allele 2) and eachof the full length heavy chain sequences are set forth below. The lightchain sequences were the same as described in Example 12.

(1) Nucleic Acid Sequence Encoding Human IgG1 Heavy Chain ConstantRegion (G1m(3) Allotype) (Allele 2) (SEQ ID NO. 207)

  1  cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 61  gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt121  ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag181  gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct241  acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca301  aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac361  cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg421  aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt481  acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca541  gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg601  agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaag accatctcca661  aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga721  tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg781  ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc841  tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc901  agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc961  agaagagcct ctccctgtct ccgggtaaat ga

(2) Protein Sequence Defining Human IgG1 Heavy Chain Constant Region(G1m(3) Allotype) (Allele 1 or 2) (SEQ ID NO. 208).

The first amino acid is derived from translation of the last nucleotideof variable region and the beginning two nucleotides of the IgG1 HeavyChain sequence.

  1  astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61  glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg121  psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn181  styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree241  mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw301  qqgnvfscsv mhealhnhyt qkslslspgk

(3) Nucleic Acid Sequence Encoding the Full Length Chain ContainingHumanized Hu2B8 Hv5-51.1 Heavy Chain Variable Region and the Human IgG1Heavy Chain Constant Region G1m(3) Allotype (Allele 2) (Signal SequenceUnderlined) (SEQ ID NO. 209)

   1  atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa  61  gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 121  tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc 181  gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat 241  ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg 301  cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt 361  ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc 421  aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 481  gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 541  ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 601  tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 661  aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 721  gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 781  ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 841  tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 901  ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 961  cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag1021  tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga agaccatctc caaagccaaa1081  gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag1141  aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag1201  tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc1261  gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg1321  aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc1381  ctctccctgt ctccgggtaa atga

(4) Protein Sequence Defining the Full Length Heavy Chain ContainingHumanized Hu2B8 Hv5-51.1 and the Human IgG1 Heavy Chain Constant RegionG1m(3) Allotype (Allele 2) (without Signal Sequence) (SEQ ID NO. 210)

  1  evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny 61  npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtivtvssas121  tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl181  yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps241  vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst301  yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt361  knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq421  gnvfscsvmh ealhnhytqk slslspgk

Each dual expression vector was transfected into 293T cells fortransient expression using DMEM 10% fetal bovine serum. Forty-eighthours after transfection, cells were washed with and then replaced withserum free medium, IS GRO™ (Irvine Scientific, Santa Ana, Calif.)containing 4 mM L-Glutamine. Supernatant was harvested daily andreplaced with fresh media for 10 days. The culture supernatants werecentrifuged, filtered (0.45 μm) and concentrated 10-100 fold. Antibodieswere purified on ProSep vA resin (Millipore), dialyzed against PBS,concentrated and sterile filtered.

Example 14 Binding Characteristics of Humanized 2B8 Variants

The humanized antibodies produced in Example 13 were characterized bytheir ability to bind hHGF and the recombinant HGF proteins produced inExample 3.

The antibodies were analyzed by surface-plasmon resonance using aBIAcore T100 instrument to assess their ability to bind hHGF and thefusion proteins discussed in Example 3. Each antibody was immobilized ona carboxymethylated dextran CM5 sensor chip (BIAcore, Catalog No.BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using astandard coupling protocol according to manufacturer's instructions.

Analyses were performed at 25° C. using PBS (GIBCO, Catalog No.14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No.R-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/mL CM-DextranSodium salt (Fluka, Catalog No. 86524) as running buffer. Supernatantcontaining different HGF fusion proteins or supernatant from cellstransfected with empty vector were injected over each antibody at a flowrate of 30 μL/min for 3 minutes. The resulting binding was determined asresonance units (RU) over baseline 30 seconds after the end ofinjection. Binding was compared to human HGF (R&D Systems, Catalog No.294-HGN-025) diluted in running buffer. Non-specific binding wasmonitored by comparing binding to a control surface. The results aresummarized in the Table 17.

TABLE 17 rhHGF (R&D rmHGF (R&D MHM chimera MHM chimera MHM chimeraAntibody Systems) Systems) (495-585) (507-585) (499-556) 2B8 Yes No YesYes Yes HE2B8-1 Yes No Yes Yes Yes HE2B8-2 Yes No Yes Yes Yes HE2B8-3Yes No Yes Yes Yes HE2B8-4 Yes No Yes Yes Yes sh2B8-9 Yes No Yes Yes Yes(G1m(3)) sh2B8-12 Yes No Yes Yes Yes (G1m(3))

The results in Table 17 demonstrate that each of the humanized 2B8-basedantibodies bind rhHGF and all three mouse-human-mouse chimeras.

Example 15 Binding Affinities of Humanized 2B8 Variants

The binding affinities and kinetics of interaction of the antibodieslisted in Table 15 were measured by surface plasmon resonance.

Mouse anti-human immunoglobulins (Jackson Labs, Catalog No. 209-005)were immobilized on carboxymethylated dextran CM4 sensor chips (BIAcore,Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No.BR-1000-50) using a standard coupling protocol according tomanufacturer's instructions. The analyses were performed at 25° C. usingPBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20(BIAcore, Catalog No. BR-1000-54), and 2 mg/mL BSA (EMD, Catalog No.2930).

The antibodies were captured in an individual flow cell at a flow rateof 10 μL/min. Injection time was variable for each antibody to yieldapproximately 20 RU of antibody captured for each cycle. Buffer or HGF(R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer wasinjected sequentially over a reference surface (no antibody captured)and the active surface (antibody to be tested) for 2 minutes at 60μL/min. The dissociation phase was monitored for 15 or 90 minutes,depending on concentration. The surface then was regenerated with 10 mMGlycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for 3minutes at a flow rate of 60 μL/min before another cycle was initiated.HGF concentrations tested were 0.46 nM to 7.5 nM.

Kinetic parameters were determined using the kinetic function of theBIAevalutation™ software with reference subtraction. Kinetic parametersfor each antibody, k_(a) (association rate constant), k_(d)(dissociation rate constant) and K_(D) (equilibrium dissociationconstant) are summarized in Table 18.

TABLE 18 Antibody k_(a) (1/Ms) kd (1/s) KD (pM) SD 2B8 1.4 × 10⁶ 1.0 ×10⁻⁵ 7.3 — HE2B8-1 2.2 × 10⁶ 1.4 × 10⁻⁵ 7.1 5.2 HE2B8-2 1.8 × 10⁶ 9.6 ×10⁻⁶ 5.2 2.7 HE2B8-3 2.0 × 10⁶ 4.1 × 10⁻⁶ 2.0 1.1 HE2B8-4 1.7 × 10⁶ 1.1× 10⁻⁵ 6.5 1.3 sh2B8-9 (G1m(17,1) 2.0 × 10⁶ 1.7 × 10⁻⁵ 8.1 5.3 sh2B8-12(G1m(17,1) 1.9 × 10⁶ 2.3 × 10⁻⁵ 12 0.4

These data show that the humanized antibodies have fast associationrates (k_(a)), very slow dissociation rates (k_(d)), and very highaffinities (K_(D)). In particular, the antibodies have affinitiesranging from 2.0-12 pM.

Example 16 Comparison of Binding Affinities at 25° C. and 37° C.

The binding affinities and kinetics of interaction of antibody HE2B8-4,sh2B8-9, sh2B8-12, and murine 2B8 were measured by surface plasmonresonance under different conditions.

Mouse anti-human immunoglobulins (Jackson Labs, Catalog No. 209-005) orrabbit anti-mouse immunoglobulins (BIAcore, Catalog No. BR-1005-14) wereimmobilized on carboxymethylated dextran CM4 sensor chips (BIAcore,Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No.BR-1000-50) using a standard coupling protocol according tomanufacturer's instructions. In the case of 25° C. measurements forsh2b8-9 and sh2B8-12, a CM5 sensor chip (BIAcore, Catalog No.BR-1006-68) was used. The analyses were performed at 25° C. and 37° C.using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20(BIAcore, Catalog No. BR-1000-54), and 2 mg/mL BSA (EMD, Catalog No.2930) as running buffer.

The antibodies were captured in an individual flow cell at a flow rateof 10 μL/min. Injection time was variable for each antibody to yieldapproximately 20 RU of antibody captured for each cycle. Buffer or HGF(R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer wasinjected sequentially over a reference surface (no antibody captured)and the active surface (antibody to be tested) for 2 minutes at 60μL/min. The dissociation phase was monitored for 15 or 90 minutes,depending on concentration. The surface of mouse anti-humanimmunoglobulins sensor chips was then regenerated with 10 mMGlycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for 3minutes at a flow rate of 60 μL/min before another cycle was initiated.The surface of rabbit anti-mouse immunoglobulins sensor chips wasregenerated with 10 mM Glycine-HCl, pH 1.7 (BIAcore, Catalog No.BR-1003-54) injected for 3 minutes at a flow rate of 60 μL/min beforeanother cycle was initiated. HGF concentrations tested were 0.46 nM to7.5nM.

Kinetic parameters were determined using the kinetic function of theBIAevaluation software with reference subtraction. Kinetic parametersfor each antibody, k_(a) (association rate constant), k_(d)(dissociation rate constant) and K_(D) (equilibrium dissociationconstant) are summarized below in Table 19.

TABLE 19 Antibody Temp. (° C.) k_(a) (1/Ms) k_(d) (1/s) K_(D) (pM) 2B825 1.6 × 10⁶ 2.1 × 10⁻⁵ 13.5 2B8 37 2.8 × 10⁶ 1.3 × 10⁻⁵ 4.5 HE2B8-4 252.0 × 10⁶ 1.2 × 10⁻⁵ 5.6 HE2B8-4 37 3.1 × 10⁶ 1.0 × 10⁻⁵ 3.3 sh2B8-9 252.0 × 10⁶ 1.7 × 10⁻⁵ 8.1 (G1m(17,1)) sh2B8-9 37 2.5 × 10⁶ 1.4 × 10⁻⁵ 5.8(G1m(3)) sh2B8-12 25 1.9 × 10⁶ 2.3 × 10⁻⁵ 12.0 (G1m(17,1)) sh2B8-12 372.4 × 10⁶ 1.1 × 10⁻⁵ 4.8 (G1m(3))

As expected, the association rate constants increased with an increasein the temperature. Surprisingly, the dissociation constants did notchange significantly with a corresponding increase in temperature.Consequently, the overall equilibrium dissociation constants (K_(D))were approximately 1.4 to 3 times smaller (higher affinity) atphysiological temperature (37° C.).

Example 17 Neutralization Activity of Humanized 2B8 Variants

The antibodies described in Example 14 were characterized for theirability to (a) inhibit the binding of hHGF to c-Met, and (b) inhibit HGFstimulated BrdU incorporation in 4 MBr-5 cells.

HGF-Met Binding Inhibition Assay (Neutralization Assay) was performed asdescribed in as follows. The antibodies were tested by ELISA for theirability to inhibit hHGF binding to c-Met. Specifically, Wallac 96-wellDELFIA assay plates (Wallac Inc., Catalog No. AAAND-0001) were coatedwith 100 μL of 6.25 μg/mL HGF (R&D Systems, Catalog No. 294-HGN-025) incarbonate coating buffer (15 mM Na₂CO₃ and 34 mM NaHCO₃, pH 9.0) for 16hours at 4° C. The plates then were blocked with 200 μL of 5% non-fatdry milk in PBS for 1 hour at room temperature. The antibodies wereprepared in a separate plate by adding increasing concentrations of theantibodies under investigation (0.033-250 nM, 2-fold-serial dilution) to2 nM biotinylated c-Met in 5% non-fat dry milk in PBS. c-Met (R&DSystems, Catalog No. 358-MT/CF) is biotinylated according tomanufacturer's instruction at 10:1 biotin to c-Met ratio (Pierce,Catalog No. 21335). 100 μL of sample per well was transferred to theassay plate and incubated for 2 hours at room temperature. The resultingplates were washed three times with PBS-0.1% Tween 20, and incubated for1 hour at room temperature with Eu-labeled Streptavidin (Wallac, CatalogNo. 1244-360) diluted 1:1000 in DELFIA assay buffer (Wallac, Catalog No.4002-0010). The resulting plates were washed 3 times with DELFIA washsolution (Wallac, Catalog No. 4010-0010) and incubated with 100 μL/wellDELFIA enhancement solution (Wallac #4001-0010) for 15 minutes at roomtemperature with agitation. The plates were read on Victor³V instrument(Perkin Elmer) using the Europium method. The IC₅₀ values werecalculated using Prism.

The IC₅₀ values obtained are shown in Table 20.

TABLE 20 Antibody IC₅₀ (nM) SD 2B8 9.2 1.2 HE2B8-1 6.0 1.2 HE2B8-2 5.71.1 HE2B8-3 5.9 1.1 HE2B8-4 6.5 1.2 sh2B8-9 (G1m(3)) 4.2 — sh2B8-12(G1m(3) 6.8 —

These results from Table 20 demonstrate that the humanized antibodiestested efficiently neutralize HGF binding to c-Met.

The antibodies in Table 17 were also tested in the cell proliferationassay described in Example 7(b). The results are summarized below inTable 21.

TABLE 21 Antibody IC₅₀ (nM) SD 2B8 0.86 0.35 HE2B8-1 0.47 0.15 HE2B8-20.66 0.13 HE2B8-3 0.55 0.28 HE2B8-4 0.58 0.26 sh2B8-9 (G1m(3)) 0.52 0.11sh2B8-12 (G1m(3)) 0.81 0.22

The results from Table 21 demonstrate that all the humanized antibodiestested inhibit HGF-induced proliferation of 4 MBr-5 cells.

Example 18 Anti-Scatter Activity of Humanized 2B8 Variants

The antibodies in Table 17 were tested in the anti-scatter assaydescribed in Example 8. The results are summarized below in Table 22.

TABLE 22 Inhibition of HGF-induced MDCK Cell Scattering Antibody Trial 1Trial 2 2B8 ++ ++ HE2B8-1 ++ ++ HE2B8-2 ++ ++ HE2B8-3 ++ ++ HE2B8-4 ++++ sh2B8-9 (G1m(3)) ++ ++ sh2B8-12 (G1m(3)) ++ ++ — No Inhibition +++Very strong, nearly complete inhibition ++ Strong inhibition +Detectable inhibition

The results in Table 22 demonstrate that all the humanized antibodiestested inhibited HGF-induced scattering to the same extent as the murinemonoclonal antibody 2B8.

Example 19 Inhibition of HGF-Stimulated c-Met Phosphorylation

The antibodies in Table 17 were tested in the c-Met phosphorylationassay described in Example 9. The results are summarized below in Table23.

TABLE 23 Average of Standard Antibody Two Trials Deviation 2B8 0.91 0.02he2B8-1 0.80 0.04 he2B8-2 0.88 0.15 he2B8-3 0.79 0.05 he2B8-4 0.75 0.14sh2B8-9 (G1m(3)) 0.93 0.03 sh2B8-12 (G1m(3)) 0.81 0.07

The results in Table 23 demonstrate that all the humanized antibodiestested are potent inhibitors of HGF-induced c-Met phosphorylation inPC-3 cells.

Example 20 Tumor Inhibition in U87MG Xenograft Model

The ability of the humanized monoclonal antibodies of the invention toinhibit tumor growth was tested in an U87MG xenograft model. U87MG cells(ATCC) were expanded in culture at 37° C. in an atmosphere containing 5%CO₂ and 95% air, using a medium comprising Dulbecco's Modified Eaglemedium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin and100 μg/mL streptomycin. The cells were subcultured and maintained bydetaching the cells from the wall of the culture dish usingtrypsin-EDTA.

Near-confluent cells were collected by trypsinization and then 5×10⁶cells in 50% Matrigel (BD Biosciences; catalog no. 356237) were injectedsubcutaneously into the upper dorsal area between the shoulder blades of7-week old female ICR SCID mice (Taconic Labs). The long (L) and short(W) diameters (mm) of tumors were measured with a caliper. Tumor volume(vol.) was calculated as: volume (mm³)=L×W²/2. When the tumors grew toapproximately 200 mm³, the tumor-bearing mice were randomized into 5groups of 10 mice each. One group received PBS and one group receivedhuman IgG control. Each of the other 4 groups received one of thehumanized antibodies (HE2B8-1, HE2B8-2, HE2B8-3, and HE2B8-4). All theantibodies were dosed at 0.25 mg/kg body weight, twice per week, byintra-peritoneal injections of 5 doses. Tumor volumes and mouse bodyweights were recorded twice per week. Tumor growth inhibition wasanalyzed using Student's t-test.

The humanized antibodies tested were active in vivo. There was 57% tumorgrowth inhibition for HE2B8-1 with a p value of 0.02, 61% tumor growthinhibition for HE2B8-2 with a p value of 0.02, 85% tumor growthinhibition for HE2B8-3, with a p value of 0.0004, and 74% tumor growthinhibition for HE2B8-4 with a p value of 0.001. No significant bodyweight loss was observed.

A subsequent study was performed as described above in female NCR nudemice (Taconic Labs) bearing subcutaneous U87MG tumors inoculated in theflank. Each group (10 mice each) received one of the followingtreatments at 0.5 mg/kg: PBS vehicle control, huIgG control, HE2B8-4, orsh2B8-9. Treatment was given intra-peritoneal twice weekly for a minimumof 5 weeks. Each treatment group demonstrated similar tumor regressionwith tumor growth inhibition of 113% for sh2B8-9 and 115% for HE2B8-4,and a minimum tumor growth delay of 30 days. Both treatments werewell-tolerated with no significant body weight loss.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. An isolated antibody that binds human hepatocytegrowth factor (HGF) comprising an immunoglobulin heavy chain variableregion comprising the amino acid sequence of SEQ ID NO. 169 and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO. 179, or an antigen binding fragment of theantibody.
 2. The antibody of claim 1, wherein the antibody is amonoclonal antibody.
 3. The antibody of claim 1, wherein the antibodycomprises an immunoglobulin heavy chain sequence comprising the aminoacid sequence of SEQ ID NO. 171 and an immunoglobulin light chainsequence comprising the amino acid sequence of SEQ ID NO.
 181. 4. Theantibody of claim 3, wherein the antibody is a monoclonal antibody. 5.The antibody of claim 1, wherein the antibody comprises animmunoglobulin heavy chain sequence comprising the amino acid sequenceof SEQ ID NO. 210 and an immunoglobulin light chain sequence comprisingthe amino acid sequence of SEQ ID NO.
 181. 6. The antibody of claim 5,wherein the antibody is a monoclonal antibody.